Subtilase Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to novel subtilase variants exhibiting increased stability and optionally on par or improved wash performance. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel subtilase variants exhibiting increased stability and optionally on par or improved wash performance. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

2. Description of the Related Art

In the detergent industry, enzymes have for many decades been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, mannosidases as well as other enzymes or mixtures thereof. Commercially the most important enzymes are proteases.

An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases Everlase®, Relase®, Ovozyme®, Polarzyme®, Liquanase®, Liquanase Ultra® and Kannase® (Novozymes A/S), Purafast®, Purafect OXP®, FN3®, FN4® and Excellase® (Genencor International, Inc.). Further, a number of variants are described in the art, such as in WO2004/041979 (Novozymes A/S) which describes subtilase variants exhibiting alterations relative to the parent subtilase in e.g. wash performance, thermal stability, storage stability or catalytic activity. The variants are suitable for use in e.g. cleaning or detergent compositions.

A number of useful subtilase variants have been described many of which have provided improved activity, stability, and solubility in different detergents. For example, WO 2007/006305 describes a list of variants comprising the substitution N261 D in combination with a number of other substituents but not in combination with a modification of position 194. EP516200 describes the substitution of position 194 but not in combination with a modification in position 261.

However, various factors make further improvement of the proteases advantageous. The washing conditions such as temperature and pH changes over time and many stains are still difficult to completely remove under conventional washing conditions. Further, in wash conditions can result in inactivation of the enzymes (due to e.g. pH, temperature or chelation instability) resulting in loss of wash performance during the wash cycle. Thus despite the intensive research in protease development there remains a need for new and improved proteases that have improved stability, in particular improved in wash stability, and preferably similar or improved wash performance compared to the parent subtilase.

SUMMARY OF THE INVENTION

The present invention relates to subtilase variants having protease activity and comprises the double substitution 194P+261 D, wherein each position corresponds to the positions of the mature polypeptide of SEQ ID NO: 2. The invention further relates to subtilase variants having protease activity, comprises the double substitution 194P+261D and further comprises one or more alterations selected from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y}.

The present invention also relates to said subtilase variants having improved stability, in particular improved in wash stability, and preferably on par or improved wash performance compared to the parent or compared to a reference protease. The present invention further relates to polynucleotides encoding the subtilase variants; compositions, preferably detergent compositions, comprising a subtilase variant; use of the compositions in a cleaning process and methods for obtaining a subtilase variant and for removing a stain from a surface.

DEFINITIONS

Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Detergent component: the term “detergent component” is defined herein to mean the types of chemicals which can be used in detergent compositions. Examples of detergent components are surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers. The detergent composition may comprise of one or more of any type of detergent component.

Detergent Composition: the term “detergent composition” includes, unless otherwise indicated, all forms of detergent compositions such as gel, granulate, liquid, paste, powder, spray or tablet compositions including heavy-duty liquids (HDL), fine-fabric liquid detergents, liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations for e.g. glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; textile and laundry pre-spotters, as well as dish wash detergents such as hand dishwashing agents, light duty dishwashing agents, machine dishwashing agents; all-purpose or heavy-duty washing agents, liquid, gel or paste-form all-purpose washing agents, liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels, foam baths; metal cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types.

In addition to containing a subtilase variant of the invention, the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

Dish wash: The term “dish wash” refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.

Dish washing composition: The term “dish washing composition” refers to all forms of compositions for cleaning hard surfaces. The present invention is not restricted to any particular type of dish wash composition or any particular detergent.

Expression: The term “expression” includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

Hard surface cleaning: The term “Hard surface cleaning” is defined herein as cleaning of hard surfaces wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash). Dish washing includes but are not limited to cleaning of plates, cups, glasses, bowls, and cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics such as melamine, metals, china, glass and acrylics.

Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Improved property: The term “improved property” means a characteristic associated with a variant that is improved compared to the parent or compared to a reference protease (the reference protease is in the context of the present application the mature polypeptide of SEQ ID NO 4 corresponding to amino acids 1 to 269 of SEQ ID NO 4.), or compared to a protease having the identical amino acid sequence of said variant but not having the alterations at one or more of said specified positions. Such improved properties include, but are not limited to, chelator stability, wash performance, protease activity, thermal activity profile, thermostability, pH activity profile, pH stability, substrate/cofactor specificity, improved surface properties, substrate specificity, product specificity, increased stability or solubility in the presence of pretreated biomass, improved stability under storage conditions (storage stability), improved in wash stability and chemical stability. Preferred embodiments are improved wash performance and improved stability, preferably improved in wash stability.

Improved stability: The term “improved stability” covers all forms of improved stability, such as improved storage stability, improved pH stability, improved thermostability, improved chelator stability, improved chemical stability and improved in wash stability. A preferred embodiment is improved in wash stability. “Improved in wash stability” is defined herein as a variant subtilase displaying improved stability during the wash cycle relative to the parent subtilase (i.e. relative to a subtilase having the identical amino acid sequence of said variant but excluding the alterations in said variant), such as relative to the mature polypeptide of SEQ ID NO: 2 or relative to the mature polypeptide of SEQ ID NO: 4. Improved in wash stability (Relative In Wash Stability Improvement Factor) can be measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

Improved wash performance: The term “improved wash performance” is defined herein as a subtilase variant displaying an alteration of the wash performance relative to the parent subtilase (i.e. relative to a subtilase having the identical amino acid sequence of said variant but excluding the alterations in said variant), such as relative to the mature polypeptide of SEQ ID NO: 2 or relative to the mature polypeptide of SEQ ID NO: 4, e.g. by increased stain removal. The term “wash performance” includes wash performance in dish wash but also in laundry. The wash performance may be determined by calculating the so-called intensity value (Int) as defined in the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash in the Materials and Methods section herein.

Isolated: The term “isolated” means a substance in a form or environment which does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

Laundering: The term “laundering” relates to both household laundering and industrial laundering and means the process of treating textiles and/or fabrics with a solution containing a detergent composition of the present invention. The laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 275 of SEQ ID NO: 2 based on the SignalP prediction program (Nielsen et al., 1997, Protein Engineering 10: 1-6)] that predicts amino acids 1 to 30 of SEQ ID NO: 2 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 269 of SEQ ID NO: 4 based on the SignalP prediction program (Nielsen et al., 1997, Protein Engineering 10: 1-6)] that predicts amino acids 1 to 27 of SEQ ID NO: 4 are a signal peptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having protease activity. In one aspect, the mature polypeptide coding sequence is nucleotides 322 to 1146 of SEQ ID NO: 1 based on the SignalP prediction program (Nielsen et al., 1997, supra)] that predicts nucleotides 1 to 90 of SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 334 to 1140 of SEQ ID NO: 3 based on the SignalP prediction program (Nielsen et al., 1997, supra)] that predicts nucleotides 1 to 81 of SEQ ID NO: 3 encode a signal peptide.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Parent: The term “parent” means a protease to which an alteration is made to produce the enzyme variants of the present invention. Thus the parent is a protease having the identical amino acid sequence of said variant but not having the alterations at one or more e.g. two or more of said specified positions. It will be understood that in the present context the expression “having identical amino acid sequence” relates to 100% sequence identity. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof. In a particular embodiment the parent is a protease with at least 60% identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

Protease: The term “protease” is defined herein as an enzyme that hydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, Calif., including supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively.

Protease activity: The term “protease activity” means a proteolytic activity (EC 3.4). Proteases of the invention are endopeptidases (EC 3.4.21). There are several protease activity types: The three main activity types are: trypsin-like where there is cleavage of amide substrates following Arg or Lys at P1, chymotrypsin-like where cleavage occurs following one of the hydrophobic amino acids at P1, and elastase-like with cleavage following an Ala at P1. For purposes of the present invention, protease activity is determined according to the procedure described in “Materials and Methods” below. The subtilase variants of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, and at least 100% of the protease activity of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Stability: The term “stability” includes storage stability and stability during use, e.g. during a wash process (in wash stability) and reflects the stability of the protease variant according to the invention as a function of time e.g. how much activity is retained when the protease is kept in solution, in particular in a detergent solution. The stability is influenced by many factors e.g. pH, temperature, detergent composition e.g. amount of builder, surfactants etc. The protease stability may be measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

Stringency conditions: The different stringency conditions are defined as follows.

The term “very low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 1×SSC, 0.2% SDS at 60° C.

The term “medium stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 1×SSC, 0.2% SDS at 65° C.

The term “medium-high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 0.5×SSC, 0.2% SDS at 65° C.

The term “high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 0.3×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 0.15×SSC, 0.2% SDS at 65° C.

Substantially pure variant: The term “substantially pure variant” means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. Preferably, the variant is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation. The variants of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant by well-known recombinant methods or by classical purification methods.

Substantially pure polynucleotide: The term “substantially pure polynucleotide” means a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered polypeptide production systems. Thus, a substantially pure polynucleotide contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′- and 3′-untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, e.g., at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, and at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form.

Textile: The term “textile” means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics made of these materials such as garments, cloths and other articles). When the term fabric or garment is used it is intended to include the broader term textiles as well.

Variant: The term “variant” means a polypeptide having protease activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding one or more (e.g. several) amino acids, e.g. 1, 2, 3, 4 or 5 amino acids adjacent to and immediately following the amino acid occupying a position.

Wash performance: The term “wash performance” is used as an enzyme's ability to remove stains present on the object to be cleaned during e.g. wash, such as laundry or hard surface cleaning. The improvement in the wash performance may be quantified by calculating the so-called intensity value (Int) defined in AMSA assay, as described in Materials and Methods section.

Wild-Type subtilase: The term “wild-type subtilase” means a protease expressed by a naturally occurring organism, such as a bacterium, archaea, yeast, fungus, plant or animal found in nature. An example of a wild-type subtilase is BPN′ i.e. amino acid 1 to 275 of SEQ ID NO: 2.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another protease. The amino acid sequence of another protease is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in another protease can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ ID NO: 2 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.

Insertions: The insertion of an additional amino acid residue such as e.g. a lysine after G195 may be indicated by: Gly195GlyLys or G195GK. Alternatively insertion of an additional amino acid residue such as lysine after G195 may be indicated by: *195aK. When more than one amino acid residue is inserted, such as e.g. a Lys and Ala after G195 this may be indicated as: Gly195GlyLysAla or G195GKA. In such cases, the inserted amino acid residue(s) may also be numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s), in this example: *195aK *195bA. In the above example, the sequences 194 to 196 would thus be:

194 195 196 Savinase  A - G - L 194 195 195a 195b 196 Variant  A - G -  K - A -  L

In cases where a substitution and an insertion occur at the same position this may be indicated as S99SD+S99A or in short S99AD. The same modification may also be indicated as S99A+*99aD.

In cases where an amino acid residue identical to the existing amino acid residue is inserted it is clear that degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G195GG or *195GaG. The same actual change could just as well be indicated as A194AG or *194aG for the change from:

194 195 196 Savinase  A - G - L

To:

194 195  195a 196 Variant  A - G -  G -  L 194 194a 195  196

Such instances will be apparent to the skilled person and the indication G195GG and corresponding indications for this type of insertions are thus meant to comprise such equivalent degenerate indications.

Multiple alterations: Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively multiple alterations may be separated be space or a comma e.g. R170Y G195E or R170Y, G195E respectively.

Different alterations: Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “Arg170Tyr,Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Alternatively different alterations or optional substitutions may be indicated in brackets e.g. Arg170[Tyr, Glu] or Arg170{Tyr, Glu} or in short R170 [Y,E] or R170 {Y, E}.

Numbering of Amino Acid Positions/Residues

If nothing else is mentioned the amino acid numbering used herein correspond to that of the subtilase BPN′ (BASBPN) sequence. For further description of the BPN′ sequence, see SEQ ID NO: 2 (amino acids 1 to 275) or Siezen et al., Protein Eng. 4 (1991) 719-737.

Table 1 of WO 89/06279 shows the alignment of the mature polypeptide of the subtilase BPN′ (BASBPN) sequence (sequence c in table 1) and the mature polypeptide of subtilisin 309 from B. Lentus, also known as Savinase®, (BLSAVI) (sequence a in table 1).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that subtilase variants comprising the double substitution 194P+261D have improved stability, in particular improved in wash stability compared to the parent subtilase.

Thus in the first aspect, the invention relates to subtilase variants having protease activity, wherein the parent subtilase comprises the double substitution 194P+261D wherein each position corresponds to the position of the mature polypeptide of SEQ ID NO: 2.

In a preferred embodiment of the invention the double substitutions 194P+261D is combined with one or more alterations selected from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y}, wherein each position corresponds to the position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4.

In a further embodiment the subtilase variant comprises the double substitution A194P+N261D. The parent subtilase may be any wild type subtilase. In one aspect, the parent subtilase is amino acids 1 to 275 of SEQ ID NO: 2. In another aspect the parent subtilase is amino acids 1 to 269 of SEQ ID NO: 4.

Thus in one embodiment, the invention relates to subtilase variants having protease activity, wherein said variant comprises the double substitution 194P+261D and one or more alterations from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 and wherein subtilase variant is

-   -   a) a polypeptide that has at least 60% but less than 100%         sequence identity to the amino acid sequence of the parent         subtilase;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with:         -   (i) the mature polypeptide coding sequence of the parent             subtilase or         -   (ii) the full-length complement of (i); or     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60% but less than 100% sequence identity to the mature         polypeptide coding sequence of the parent subtilase.

In an embodiment, the subtilase variant has at least 65% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 70% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 75% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 80% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 85% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 90% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 93% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 95% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 96% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 97% but less than 100% sequence identity to the mature polypeptide of the parent subtilase. In an embodiment, the subtilase variant has at least 98% but less than 100% sequence identity to the mature polypeptide of the parent subtilase.

In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 65% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 70% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 75% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 80% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 85% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 90% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 93% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 95% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 96% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 97% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 98% but less than 100% sequence identity to the mature polypeptide coding sequence of the parent subtilase.

In one aspect, the total number of alterations compared to the parent subtilase is between 3 and 30, preferably between 3 and 20, more preferably between 3 and 15, even more preferably between 3 and 10, most preferably between 3 and 8 alterations. In another aspect, total number of alterations in the parent subtilase is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 alterations.

In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of the parent subtilase. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of the parent subtilase.

In a second embodiment, the invention relates to subtilase variants having protease activity, wherein said variant comprises the double substitution A194P+N261D and one or more alterations from the group consisting of S3{I, L, P, V, Y}, S9{A, G, M, T}, N18{A, G, M, S, T}, P40{D, E}, N43{D, E}, R45{D, E}, T58{F, Y}, Q59{D, E}, G61{D, E}, 172{L, V}, N76{D, E}, S99{A, E, G, M, T}, V104{F, Y}, S105{D, E}, H120{D, E, 1, L, V}, S130*, A133{D, E}, S141{F, Y}, Q182{D, E}, N183{D, E}, N184{D, E}, V205{I, L}, Q206{D, E}, S212E, N218{D, E}, T224{A, G, M, S}, P225{A, G, M, S, T}, A228{G, M, S, T}, Q236{D, E}, Q245{H, K}, S259{D, E} and L262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 and wherein subtilase variant is

-   -   a) a polypeptide that has at least 60% but less than 100%         sequence identity to the mature polypeptide of SEQ ID NO: 4;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with:         -   (i) the mature polypeptide coding sequence of SEQ ID NO: 3             or         -   (ii) the full-length complement of (i); and     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60% but less than 100% sequence identity to the mature         polypeptide coding sequence of SEQ ID NO: 3.

In an embodiment, the subtilase variant has at least 65% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 70% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 75% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 80% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 85% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 90% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 93% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 95% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 96% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 97% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. In an embodiment, the subtilase variant has at least 98% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4.

In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 65% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 70% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 75% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 80% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 85% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 90% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 93% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 95% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 96% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 97% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3. In an embodiment, the subtilase variant is encoded by a polynucleotide that has at least 98% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3.

In one aspect, the total number of alterations in the mature polypeptide of SEQ ID NO: 4 is between 3 and 30, preferably between 3 and 20, more preferably between 3 and 15, even more preferably between 3 and 10, most preferably between 3 and 8 alterations. In another aspect, total number of alterations in the mature polypeptide of SEQ ID NO: 4 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 alterations.

In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of SEQ ID NO: 4. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of SEQ ID NO: 4.

A preferred embodiment of the invention relates to subtilase variants having protease activity, wherein said variant comprises the double substitution 194P+261D and one or more alterations from the group consisting of 3V, 3Y, 9M, 18S, 18T, 18D, 40E, 43D, 43E, 45E, 58Y, 59E, 61D, 72V, 76D, 99D, 104Y, 1054D, 120I, 120D, 130*, 133E, 141F, 182E, 183D, 184D, 205I, 212D, 218D, 228S, 236E and 262Y wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 and wherein subtilase variant is

-   -   a) a polypeptide that has at least 60% but less than 100%         sequence identity to the amino acid sequence of the parent         subtilase;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with:         -   (i) the mature polypeptide coding sequence of the parent             subtilase or         -   (ii) the full-length complement of (i); or     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60% but less than 100% sequence identity to the mature         polypeptide coding sequence of the parent subtilase.

Another preferred embodiment of the invention relates to subtilase variants having protease activity, wherein said variant comprises the double substitution 194P+261D and one or more alterations from the group consisting of 3V, 3Y, 9M, 18S, 18T, 18D, 40E, 43D, 43E, 45E, 58Y, 59E, 61D, 72V, 76D, 99D, 104Y, 1054D, 120I, 120D, 130*, 133E, 141F, 182E, 183D, 184D, 205I, 212D, 218D, 228S, 236E and 262Y wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 and wherein subtilase variant is

-   -   a) a polypeptide that has at least 60% but less than 100%         sequence identity to the amino acid sequence of SEQ ID NO 4;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with:         -   (i) the mature polypeptide coding sequence of SEQ ID NO 1 or         -   (ii) the full-length complement of (i); or     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60% but less than 100% sequence identity to the mature         polypeptide coding sequence of SEQ ID NO 4.

Variants

In one aspect of the invention, the subtilase variant comprises or consists of one or more of the alterations in table 1, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2. In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4.

TABLE 1 Subtilase Variants 3F + 194P + 261D 76D + 194P + 261D 194P + 206E + 261D 3I + 194P + 261D 76E + 194P + 261D 194P + 212D + 261D 3L + 194P + 261D 99A + 194P + 261D 194P + 212E + 261D 3V + 194P + 261D 99D + 194P + 261D 194P + 218D + 261D 3Y + 194P + 261D 99E + 194P + 261D 194P + 218E + 261D 9A + 194P + 261D 99M + 194P + 261D 194P + 224A + 261D 9G + 194P + 261D 99T + 194P + 261D 194P + 224G + 261D 9M + 194P + 261D 104F + 194P + 261D 194P + 224M + 261D 9T + 194P + 261D 104Y + 194P + 261D 194P + 224S + 261D 18A + 194P + 261D 105D + 194P + 261D 194P + 225A + 261D 18D + 194P + 261D 105E + 194P + 261D 194P + 225G + 261D 18E + 194P + 261D 120D + 194P + 261D 194P + 225M + 261D 18G + 194P + 261D 120E + 194P + 261D 194P + 225S + 261D 18M + 194P + 261D 120I + 194P + 261D 194P + 225T + 261D 18S + 194P + 261D 120L + 194P + 261D 194P + 228G + 261D 18T + 194P + 261D 120V + 194P + 261D 194P + 228M + 261D 40D + 194P + 261D 130* + 194P + 261D 194P + 228S + 261D 40E + 194P + 261D 133D + 194P + 261D 194P + 228T + 261D 43D + 194P + 261D 133E + 194P + 261D 194P + 236D + 261D 43E + 194P + 261D 141F + 194P + 261D 194P + 236E + 261D 45D + 194P + 261D 141Y + 194P + 261D 194P + 245H + 261D 45E + 194P + 261D 182D + 194P + 261D 194P + 245K + 261D 58F + 194P + 261D 182E + 194P + 261D 194P + 259D + 261D 58Y + 194P + 261D 183D + 194P + 261D 194P + 259E + 261D 59D + 194P + 261D 183E + 194P + 261D 194P + 261D + 262F 59E + 194P + 261D 184D + 194P + 261D 194P + 261D + 262W 61D + 194P + 261D 184E + 194P + 261D 194P + 261D + 262Y 61E + 194P + 261D 194P + 205I + 261D 72L + 194P + 261D 194P + 205L + 261D 72V + 194P + 261D 194P + 206D + 261D

In one embodiment, the subtilase variant comprises or consists of one or more of the alterations described in table 1 in the mature polypeptide of SEQ ID NO: 2. In another embodiment, the subtilase variant comprises or consists of one or more of the alterations described in table 1 in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S3F+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S3I+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S3L+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S3V+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S3Y+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S9A+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S9G+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S9M+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S9T+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18A+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18G+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18M+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18S+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N18T+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations P40D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations P40E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N43D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N43E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations R45D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations R45E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations T58F+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations T58Y+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations Q59D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations Q59E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations G61D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations G61E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations I72L+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations I72V+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N76D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N76E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S99A+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S99D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S99E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S99M+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S99T+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations V104F+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations V104Y+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S105D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S105E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations H120D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations H120E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations H120I+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations H120L+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations H120V+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S130*+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A133D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A133E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S141F+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations S141Y+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations Q182D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations Q182E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N183D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N183E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N184D+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations N184E+A194P+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+V205I+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+V205L+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+Q206D+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+Q206E+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+S212D+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+S212E+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+N218D+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+N218E+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+T224A+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+T224G+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+T224M+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+T224S+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+P225A+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+P225G+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+P225M+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+P225S+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+P225T+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+A228G+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+A228M+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+A228S+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+A228T+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+Q236D+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+Q236E+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+Q245H+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+Q245K+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+S259D+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+S259E+N261D in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+N261D+L262F in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+N261D+L262W in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant comprises or consists of the alterations A194P+N261D+L262Y in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4.

The subtilase variants may further comprise a substitution at one or more positions (e.g. several) selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 146, 188, 217, 245, 255 and 270, preferably positions 15, 63, 68 and/or 217 (numbering according to SEQ ID NO: 2). It will be clear to the skilled artisan that if a position has already been altered once, then it will not be altered a second time. In a one embodiment the subtilase variant further comprises a deletion of the amino acid at position 132, or an insertion of an additional amino acid at position 99 (numbering according to SEQ ID NO: 2). In a more preferred embodiment, the subtilase variant further comprises one or more substitutions selected from the group consisting of 4I, 9{H, K, R}, 12{D, E}, 14T, 15{G, M, S, T}, 40{D, E}, 63G, 68{A, G, 1, L, M, S, T}, 79T, 86H, 88V, 92S, 98T, 101L, 146S, 188{A, G, M, T}, 217L, 245R and/or 270{G, M, S, T} (numbering according to SEQ ID NO: 2). In an even more preferred embodiment, the subtilase variant further comprises one or more substitutions selected from the group consisting of V4I, S9R, Q12E, P14T, A15T, P40E, S63G, V68A, I79T, P86H, A88V, A92S, A98T, S101L, G146S, S188T, Y217L, Q245R, and/or A270G and/or a deletion in position S132* in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

Thus in further embodiments of the invention, the subtilase variant comprises or consists of one of the alterations in table 2 in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2. In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4.

TABLE 2 Subtilase Variants S3I + S9A + A194P + N261D S3L + G61D + A194P + V205I + N261D S3L + S9A + A194P + N261D S3F + G61D + A194P + V205I + N261D S3F + S9A + A194P + N261D S3V + G61D + A194P + V205I + N261D S3V + S9A + A194P + N261D S3Y + G61D + A194P + V205I + N261D S3Y + S9A + A194P + N261D S3I + G61E + A194P + V205I + N261D S3I + S9G + A194P + N261D S3L + G61E + A194P + V205I + N261D S3L + S9G + A194P + N261D S3F + G61E + A194P + V205I + N261D S3F + S9G + A194P + N261D S3V + G61E + A194P + V205I + N261D S3V + S9G + A194P + N261D S3Y + G61E + A194P + V205I + N261D S3Y + S9G + A194P + N261D S3I + G61D + A194P + V205L + N261D S3I + S9M + A194P + N261D S3L + G61D + A194P + V205L + N261D S3L + S9M + A194P + N261D S3F + G61D + A194P + V205L + N261D S3F + S9M + A194P + N261D S3V + G61D + A194P + V205L + N261D S3V + S9M + A194P + N261D S3Y + G61D + A194P + V205L + N261D S3Y + S9M + A194P + N261D S3I + G61E + A194P + V205L + N261D S3I + S9R + A194P + N261D S3L + G61E + A194P + V205L + N261D S3L + S9R + A194P + N261D S3F + G61E + A194P + V205L + N261D S3F + S9R + A194P + N261D S3V + G61E + A194P + V205L + N261D S3V + S9R + A194P + N261D S3Y + G61E + A194P + V205L + N261D S3Y + S9R + A194P + N261D S3I + G61D + A194P + Q245H + N261D S3I + S9T + A194P + N261D S3L + G61D + A194P + Q245H + N261D S3L + S9T + A194P + N261D S3F + G61D + A194P + Q245H + N261D S3F + S9T + A194P + N261D S3V + G61D + A194P + Q245H + N261D S3V + S9T + A194P + N261D S3Y + G61D + A194P + Q245H + N261D S3Y + S9T + A194P + N261D S3I + G61E + A194P + Q245H + N261D S3I + N43D + A194P + N261D S3L + G61E + A194P + Q245H + N261D S3L + N43D + A194P + N261D S3F + G61E + A194P + Q245H + N261D S3F + N43D + A194P + N261D S3V + G61E + A194P + Q245H + N261D S3V + N43D + A194P + N261D S3Y + G61E + A194P + Q245H + N261D S3Y + N43D + A194P + N261D S3I + G61D + A194P + Q245K + N261D S3I + N43E + A194P + N261D S3L + G61D + A194P + Q245K + N261D S3L + N43E + A194P + N261D S3F + G61D + A194P + Q245K + N261D S3F + N43E + A194P + N261D S3V + G61D + A194P + Q245K + N261D S3V + N43E + A194P + N261D S3Y + G61D + A194P + Q245K + N261D S3Y + N43E + A194P + N261D S3I + G61E + A194P + Q245K + N261D S3I + G61D + A194P + N261D S3L + G61E + A194P + Q245K + N261D S3L + G61D + A194P + N261D S3F + G61E + A194P + Q245K + N261D S3F + G61D + A194P + N261D S3V + G61E + A194P + Q245K + N261D S3V + G61D + A194P + N261D S3Y + G61E + A194P + Q245K + N261D S3Y + G61D + A194P + N261D S3I + G61D + A194P + Q245R + N261D S3I + G61E + A194P + N261D S3L + G61D + A194P + Q245R + N261D S3L + G61E + A194P + N261D S3F + G61D + A194P + Q245R + N261D S3F + G61E + A194P + N261D S3V + G61D + A194P + Q245R + N261D S3V + G61E + A194P + N261D S3Y + G61D + A194P + Q245R + N261D S3Y + G61E + A194P + N261D S3I + G61E + A194P + Q245R + N261D S3I + A194P + V205I + N261D S3L + G61E + A194P + Q245R + N261D S3L + A194P + V205I + N261D S3F + G61E + A194P + Q245R + N261D S3F + A194P + V205I + N261D S3V + G61E + A194P + Q245R + N261D S3V + A194P + V205I + N261D S3Y + G61E + A194P + Q245R + N261D S3Y + A194P + V205I + N261D S3I + G61D + A194P + N261D + L262F S3I + A194P + V205L + N261D S3L + G61D + A194P + N261D + L262F S3L + A194P + V205L + N261D S3F + G61D + A194P + N261D + L262F S3F + A194P + V205L + N261D S3V + G61D + A194P + N261D + L262F S3V + A194P + V205L + N261D S3Y + G61D + A194P + N261D + L262F S3Y + A194P + V205L + N261D S3I + G61E + A194P + N261D + L262F S3I + A194P + Q245H + N261D S3L + G61E + A194P + N261D + L262F S3L + A194P + Q245H + N261D S3F + G61E + A194P + N261D + L262F S3F + A194P + Q245H + N261D S3V + G61E + A194P + N261D + L262F S3V + A194P + Q245H + N261D S3Y + G61E + A194P + N261D + L262F S3Y + A194P + Q245H + N261D S3I + G61D + A194P + N261D + L262W S3I + A194P + Q245R + N261D S3L + G61D + A194P + N261D + L262W S3L + A194P + Q245R + N261D S3F + G61D + A194P + N261D + L262W S3F + A194P + Q245R + N261D S3V + G61D + A194P + N261D + L262W S3V + A194P + Q245R + N261D S3Y + G61D + A194P + N261D + L262W S3Y + A194P + Q245R + N261D S3I + G61E + A194P + N261D + L262W S3I + A194P + Q245T + N261D S3L + G61E + A194P + N261D + L262W S3L + A194P + Q245T + N261D S3F + G61E + A194P + N261D + L262W S3F + A194P + Q245T + N261D S3V + G61E + A194P + N261D + L262W S3V + A194P + Q245T + N261D S3Y + G61E + A194P + N261D + L262W S3Y + A194P + Q245T + N261D S3I + G61D + A194P + N261D + L262Y S3I + A194P + N261D + L262F S3L + G61D + A194P + N261D + L262Y S3L + A194P + N261D + L262F S3F + G61D + A194P + N261D + L262Y S3F + A194P + N261D + L262F S3V + G61D + A194P + N261D + L262Y S3V + A194P + N261D + L262F S3Y + G61D + A194P + N261D + L262Y S3Y + A194P + N261D + L262F S3I + G61E + A194P + N261D + L262Y S3I + A194P + N261D + L262W S3L + G61E + A194P + N261D + L262Y S3L + A194P + N261D + L262W S3F + G61E + A194P + N261D + L262Y S3F + A194P + N261D + L262W S3V + G61E + A194P + N261D + L262Y S3V + A194P + N261D + L262W S3Y + G61E + A194P + N261D + L262Y S3Y + A194P + N261D + L262W S3I + A194P + V205I + Q245H + N261D S3I + A194P + N261D + L262Y S3L + A194P + V205I + Q245H + N261D S3L + A194P + N261D + L262Y S3F + A194P + V205I + Q245H + N261D S3F + A194P + N261D + L262Y S3V + A194P + V205I + Q245H + N261D S3V + A194P + N261D + L262Y S3Y + A194P + V205I + Q245H + N261D S3Y + A194P + N261D + L262Y S3I + A194P + V205L + Q245H + N261D S9A + N43D + A194P + N261D S3L + A194P + V205L + Q245H + N261D S9G + N43D + A194P + N261D S3F + A194P + V205L + Q245H + N261D S9M + N43D + A194P + N261D S3V + A194P + V205L + Q245H + N261D S9R + N43D + A194P + N261D S3Y + A194P + V205L + Q245H + N261D S9T + N43D + A194P + N261D S3I + A194P + V205I + Q245K + N261D S9A + N43E + A194P + N261D S3L + A194P + V205I + Q245K + N261D S9G + N43E + A194P + N261D S3F + A194P + V205I + Q245K + N261D S9M + N43E + A194P + N261D S3V + A194P + V205I + Q245K + N261D S9R + N43E + A194P + N261D S3Y + A194P + V205I + Q245K + N261D S9T + N43E + A194P + N261D S3I + A194P + V205L + Q245K + N261D S9A + G61D + A194P + N261D S3L + A194P + V205L + Q245K + N261D S9G + G61D + A194P + N261D S3F + A194P + V205L + Q245K + N261D S9M + G61D + A194P + N261D S3V + A194P + V205L + Q245K + N261D S9R + G61D + A194P + N261D S3Y + A194P + V205L + Q245K + N261D S9T + G61D + A194P + N261D S3I + A194P + V205I + Q245R + N261D S9A + G61E + A194P + N261D S3L + A194P + V205I + Q245R + N261D S9G + G61E + A194P + N261D S3F + A194P + V205I + Q245R + N261D S9M + G61E + A194P + N261D S3V + A194P + V205I + Q245R + N261D S9R + G61E + A194P + N261D S3Y + A194P + V205I + Q245R + N261D S9T + G61E + A194P + N261D S3I + A194P + V205L + Q245R + N261D S9A + A194P + V205I + N261D S3L + A194P + V205L + Q245R + N261D S9G + A194P + V205I + N261D S3F + A194P + V205L + Q245R + N261D S9M + A194P + V205I + N261D S3V + A194P + V205L + Q245R + N261D S9R + A194P + V205I + N261D S3Y + A194P + V205L + Q245R + N261D S9T + A194P + V205I + N261D S3I + A194P + V205I + N261 + L262F S9A + A194P + V205L + N261D S3L + A194P + V205I + N261 + L262F S9G + A194P + V205L + N261D S3F + A194P + V205I + N261 + L262F S9M + A194P + V205L + N261D S3V + A194P + V205I + N261 + L262F S9R + A194P + V205L + N261D S3Y + A194P + V205I + N261 + L262F S9T + A194P + V205L + N261D S3I + A194P + V205L + N261 + L262F S9A + A194P + Q245H + N261D S3L + A194P + V205L + N261 + L262F S9G + A194P + Q245H + N261D S3F + A194P + V205L + N261 + L262F S9M + A194P + Q245H + N261D S3V + A194P + V205L + N261 + L262F S9R + A194P + Q245H + N261D S3Y + A194P + V205L + N261 + L262F S9T + A194P + Q245H + N261D S3I + A194P + V205I + N261 + L262W S9A + A194P + Q245R + N261D S3L + A194P + V205I + N261 + L262W S9G + A194P + Q245R + N261D S3F + A194P + V205I + N261 + L262W S9M + A194P + Q245R + N261D S3V + A194P + V205I + N261 + L262W S9R + A194P + Q245R + N261D S3Y + A194P + V205I + N261 + L262W S9T + A194P + Q245R + N261D S3I + A194P + V205L + N261 + L262W S9A + A194P + Q245T + N261D S3L + A194P + V205L + N261 + L262W S9G + A194P + Q245T + N261D S3F + A194P + V205L + N261 + L262W S9M + A194P + Q245T + N261D S3V + A194P + V205L + N261 + L262W S9R + A194P + Q245T + N261D S3Y + A194P + V205L + N261 + L262W S9T + A194P + Q245T + N261D S3I + A194P + V205I + N261 + L262Y S9A + A194P + N261D + L262F S3L + A194P + V205I + N261 + L262Y S9G + A194P + N261D + L262F S3F + A194P + V205I + N261 + L262Y S9M + A194P + N261D + L262F S3V + A194P + V205I + N261 + L262Y S9R + A194P + N261D + L262F S3Y + A194P + V205I + N261 + L262Y S9T + A194P + N261D + L262F S3I + A194P + V205L + N261 + L262Y S9A + A194P + N261D + L262W S3L + A194P + V205L + N261 + L262Y S9G + A194P + N261D + L262W S3F + A194P + V205L + N261 + L262Y S9M + A194P + N261D + L262W S3V + A194P + V205L + N261 + L262Y S9R + A194P + N261D + L262W S3Y + A194P + V205L + N261 + L262Y S9T + A194P + N261D + L262W S3I + A194P + Q245H + N261 + L262F S9A + A194P + N261D + L262Y S3L + A194P + Q245H + N261 + L262F S9G + A194P + N261D + L262Y S3F + A194P + Q245H + N261 + L262F S9M + A194P + N261D + L262Y S3V + A194P + Q245H + N261 + L262F S9R + A194P + N261D + L262Y S3Y + A194P + Q245H + N261 + L262F S9T + A194P + N261D + L262Y S3I + A194P + Q245R + N261 + L262F N43D + G61D + A194P + N261D S3L + A194P + Q245R + N261 + L262F N43E + G61D + A194P + N261D S3F + A194P + Q245R + N261 + L262F N43D + G61E + A194P + N261D S3V + A194P + Q245R + N261 + L262F N43E + G61E + A194P + N261D S3Y + A194P + Q245R + N261 + L262F N43D + A194P + V205I + N261D S3I + A194P + Q245T + N261 + L262F N43E + A194P + V205I + N261D S3L + A194P + Q245T + N261 + L262F N43D + A194P + V205L + N261D S3F + A194P + Q245T + N261 + L262F N43E + A194P + V205L + N261D S3V + A194P + Q245T + N261 + L262F N43D + A194P + Q245H + N261D S3Y + A194P + Q245T + N261 + L262F N43E + A194P + Q245H + N261D S3I + A194P + Q245H + N261 + L262W N43D + A194P + Q245R + N261D S3L + A194P + Q245H + N261 + L262W N43E + A194P + Q245R + N261D S3F + A194P + Q245H + N261 + L262W N43D + A194P + Q245T + N261D S3V + A194P + Q245H + N261 + L262W N43E + A194P + Q245T + N261D S3Y + A194P + Q245H + N261 + L262W N43D + A194P + N261D + L262F S3I + A194P + Q245R + N261 + L262W N43E + A194P + N261D + L262F S3L + A194P + Q245R + N261 + L262W N43D + A194P + N261D + L262W S3F + A194P + Q245R + N261 + L262W N43E + A194P + N261D + L262W S3V + A194P + Q245R + N261 + L262W N43D + A194P + N261D + L262Y S3Y + A194P + Q245R + N261 + L262W N43E + A194P + N261D + L262Y S3I + A194P + Q245T + N261 + L262W G61D + A194P + V205I + N261D S3L + A194P + Q245T + N261 + L262W G61E + A194P + V205I + N261D S3F + A194P + Q245T + N261 + L262W G61D + A194P + V205L + N261D S3V + A194P + Q245T + N261 + L262W G61E + A194P + V205L + N261D S3Y + A194P + Q245T + N261 + L262W G61D + A194P + Q245H + N261D S3I + A194P + Q245H + N261 + L262Y G61E + A194P + Q245H + N261D S3L + A194P + Q245H + N261 + L262Y G61D + A194P + Q245R + N261D S3F + A194P + Q245H + N261 + L262Y G61E + A194P + Q245R + N261D S3V + A194P + Q245H + N261 + L262Y G61D + A194P + Q245T + N261D S3Y + A194P + Q245H + N261 + L262Y G61E + A194P + Q245T + N261D S3I + A194P + Q245R + N261 + L262Y G61D + A194P + N261D + L262F S3L + A194P + Q245R + N261 + L262Y G61E + A194P + N261D + L262F S3F + A194P + Q245R + N261 + L262Y G61D + A194P + N261D + L262W S3V + A194P + Q245R + N261 + L262Y G61E + A194P + N261D + L262W S3Y + A194P + Q245R + N261 + L262Y G61D + A194P + N261D + L262Y S3I + A194P + Q245T + N261 + L262Y G61E + A194P + N261D + L262Y S3L + A194P + Q245T + N261 + L262Y A194P + V205I + Q245H + N261D S3F + A194P + Q245T + N261 + L262Y A194P + V205L + Q245H + N261D S3V + A194P + Q245T + N261 + L262Y A194P + V205I + Q245R + N261D S3Y + A194P + Q245T + N261 + L262Y A194P + V205L + Q245R + N261D S9A + N43D + G61D + A194P + N261D A194P + V205I + Q245T + N261D S9G + N43D + G61D + A194P + N261D A194P + V205L + Q245T + N261D S9M + N43D + G61D + A194P + N261D A194P + V205I + N261D + L262F S9R + N43D + G61D + A194P + N261D A194P + V205L + N261D + L262F S9T + N43D + G61D + A194P + N261D A194P + V205I + N261D + L262W S9A + N43E + G61D + A194P + N261D A194P + V205L + N261D + L262W S9G + N43E + G61D + A194P + N261D A194P + V205I + N261D + L262Y S9M + N43E + G61D + A194P + N261D A194P + V205L + N261D + L262Y S9R + N43E + G61D + A194P + N261D A194P + Q245H + N261D + L262F S9T + N43E + G61D + A194P + N261D A194P + Q245K + N261D + L262F S9A + N43D + G61E + A194P + N261D A194P + Q245R + N261D + L262F S9G + N43D + G61E + A194P + N261D A194P + Q245H + N261D + L262W S9M + N43D + G61E + A194P + N261D A194P + Q245K + N261D + L262W S9R + N43D + G61E + A194P + N261D A194P + Q245R + N261D + L262W S9T + N43D + G61E + A194P + N261D A194P + Q245H + N261D + L262Y S9A + N43E + G61E + A194P + N261D A194P + Q245K + N261D + L262Y S9G + N43E + G61E + A194P + N261D A194P + Q245R + N261D + L262Y S9M + N43E + G61E + A194P + N261D S3I + S9A + N43D + A194P + N261D S9R + N43E + G61E + A194P + N261D S3L + S9A + N43D + A194P + N261D S9T + N43E + G61E + A194P + N261D S3F + S9A + N43D + A194P + N261D S9A + N43D + A194P + V205I + N261D S3V + S9A + N43D + A194P + N261D S9G + N43D + A194P + V205I + N261D S3Y + S9A + N43D + A194P + N261D S9M + N43D + A194P + V205I + N261D S3I + S9G + N43D + A194P + N261D S9R + N43D + A194P + V205I + N261D S3L + S9G + N43D + A194P + N261D S9T + N43D + A194P + V205I + N261D S3F + S9G + N43D + A194P + N261D S9A + N43E + A194P + V205I + N261D S3V + S9G + N43D + A194P + N261D S9G + N43E + A194P + V205I + N261D S3Y + S9G + N43D + A194P + N261D S9M + N43E + A194P + V205I + N261D S3I + S9M + N43D + A194P + N261D S9R + N43E + A194P + V205I + N261D S3L + S9M + N43D + A194P + N261D S9T + N43E + A194P + V205I + N261D S3F + S9M + N43D + A194P + N261D S9A + N43D + A194P + V205L + N261D S3V + S9M + N43D + A194P + N261D S9G + N43D + A194P + V205L + N261D S3Y + S9M + N43D + A194P + N261D S9M + N43D + A194P + V205L + N261D S3I + S9R + N43D + A194P + N261D S9R + N43D + A194P + V205L + N261D S3L + S9R + N43D + A194P + N261D S9T + N43D + A194P + V205L + N261D S3F + S9R + N43D + A194P + N261D S9A + N43E + A194P + V205L + N261D S3V + S9R + N43D + A194P + N261D S9G + N43E + A194P + V205L + N261D S3Y + S9R + N43D + A194P + N261D S9M + N43E + A194P + V205L + N261D S3I + S9T + N43D + A194P + N261D S9R + N43E + A194P + V205L + N261D S3L + S9T + N43D + A194P + N261D S9T + N43E + A194P + V205L + N261D S3F + S9T + N43D + A194P + N261D S9A + N43D + A194P + Q245H + N261D S3V + S9T + N43D + A194P + N261D S9G + N43D + A194P + Q245H + N261D S3Y + S9T + N43D + A194P + N261D S9M + N43D + A194P + Q245H + N261D S3I + S9A + N43E + A194P + N261D S9R + N43D + A194P + Q245H + N261D S3L + S9A + N43E + A194P + N261D S9T + N43D + A194P + Q245H + N261D S3F + S9A + N43E + A194P + N261D S9A + N43E + A194P + Q245H + N261D S3V + S9A + N43E + A194P + N261D S9G + N43E + A194P + Q245H + N261D S3Y + S9A + N43E + A194P + N261D S9M + N43E + A194P + Q245H + N261D S3I + S9G + N43E + A194P + N261D S9R + N43E + A194P + Q245H + N261D S3L + S9G + N43E + A194P + N261D S9T + N43E + A194P + Q245H + N261D S3F + S9G + N43E + A194P + N261D S9A + N43D + A194P + Q245K + N261D S3V + S9G + N43E + A194P + N261D S9G + N43D + A194P + Q245K + N261D S3Y + S9G + N43E + A194P + N261D S9M + N43D + A194P + Q245K + N261D S3I + S9M + N43E + A194P + N261D S9R + N43D + A194P + Q245K + N261D S3L + S9M + N43E + A194P + N261D S9T + N43D + A194P + Q245K + N261D S3F + S9M + N43E + A194P + N261D S9A + N43E + A194P + Q245K + N261D S3V + S9M + N43E + A194P + N261D S9G + N43E + A194P + Q245K + N261D S3Y + S9M + N43E + A194P + N261D S9M + N43E + A194P + Q245K + N261D S3I + S9R + N43E + A194P + N261D S9R + N43E + A194P + Q245K + N261D S3L + S9R + N43E + A194P + N261D S9T + N43E + A194P + Q245K + N261D S3F + S9R + N43E + A194P + N261D S9A + N43D + A194P + Q245R + N261D S3V + S9R + N43E + A194P + N261D S9G + N43D + A194P + Q245R + N261D S3Y + S9R + N43E + A194P + N261D S9M + N43D + A194P + Q245R + N261D S3I + S9T + N43E + A194P + N261D S9R + N43D + A194P + Q245R + N261D S3L + S9T + N43E + A194P + N261D S9T + N43D + A194P + Q245R + N261D S3F + S9T + N43E + A194P + N261D S9A + N43E + A194P + Q245R + N261D S3V + S9T + N43E + A194P + N261D S9G + N43E + A194P + Q245R + N261D S3Y + S9T + N43E + A194P + N261D S9M + N43E + A194P + Q245R + N261D S3I + S9A + G61D + A194P + N261D S9R + N43E + A194P + Q245R + N261D S3L + S9A + G61D + A194P + N261D S9T + N43E + A194P + Q245R + N261D S3F + S9A + G61D + A194P + N261D S9A + N43D + A194P + N261 + L262F S3V + S9A + G61D + A194P + N261D S9G + N43D + A194P + N261 + L262F S3Y + S9A + G61D + A194P + N261D S9M + N43D + A194P + N261 + L262F S3I + S9G + G61D + A194P + N261D S9R + N43D + A194P + N261 + L262F S3L + S9G + G61D + A194P + N261D S9T + N43D + A194P + N261 + L262F S3F + S9G + G61D + A194P + N261D S9A + N43E + A194P + N261 + L262F S3V + S9G + G61D + A194P + N261D S9G + N43E + A194P + N261 + L262F S3Y + S9G + G61D + A194P + N261D S9M + N43E + A194P + N261 + L262F S3I + S9M + G61D + A194P + N261D S9R + N43E + A194P + N261 + L262F S3L + S9M + G61D + A194P + N261D S9T + N43E + A194P + N261 + L262F S3F + S9M + G61D + A194P + N261D S9A + N43D + A194P + N261 + L262W S3V + S9M + G61D + A194P + N261D S9G + N43D + A194P + N261 + L262W S3Y + S9M + G61D + A194P + N261D S9M + N43D + A194P + N261 + L262W S3I + S9R + G61D + A194P + N261D S9R + N43D + A194P + N261 + L262W S3L + S9R + G61D + A194P + N261D S9T + N43D + A194P + N261 + L262W S3F + S9R + G61D + A194P + N261D S9A + N43E + A194P + N261 + L262W S3V + S9R + G61D + A194P + N261D S9G + N43E + A194P + N261 + L262W S3Y + S9R + G61D + A194P + N261D S9M + N43E + A194P + N261 + L262W S3I + S9T + G61D + A194P + N261D S9R + N43E + A194P + N261 + L262W S3L + S9T + G61D + A194P + N261D S9T + N43E + A194P + N261 + L262W S3F + S9T + G61D + A194P + N261D S9A + N43D + A194P + N261 + L262Y S3V + S9T + G61D + A194P + N261D S9G + N43D + A194P + N261 + L262Y S3Y + S9T + G61D + A194P + N261D S9M + N43D + A194P + N261 + L262Y S3I + S9A + G61E + A194P + N261D S9R + N43D + A194P + N261 + L262Y S3L + S9A + G61E + A194P + N261D S9T + N43D + A194P + N261 + L262Y S3F + S9A + G61E + A194P + N261D S9A + N43E + A194P + N261 + L262Y S3V + S9A + G61E + A194P + N261D S9G + N43E + A194P + N261 + L262Y S3Y + S9A + G61E + A194P + N261D S9M + N43E + A194P + N261 + L262Y S3I + S9G + G61E + A194P + N261D S9R + N43E + A194P + N261 + L262Y S3L + S9G + G61E + A194P + N261D S9T + N43E + A194P + N261 + L262Y S3F + S9G + G61E + A194P + N261D S9A + G61D + A194P + V205I + N261D S3V + S9G + G61E + A194P + N261D S9G + G61D + A194P + V205I + N261D S3Y + S9G + G61E + A194P + N261D S9M + G61D + A194P + V205I + N261D S3I + S9M + G61E + A194P + N261D S9R + G61D + A194P + V205I + N261D S3L + S9M + G61E + A194P + N261D S9T + G61D + A194P + V205I + N261D S3F + S9M + G61E + A194P + N261D S9A + G61E + A194P + V205I + N261D S3V + S9M + G61E + A194P + N261D S9G + G61E + A194P + V205I + N261D S3Y + S9M + G61E + A194P + N261D S9M + G61E + A194P + V205I + N261D S3I + S9R + G61E + A194P + N261D S9R + G61E + A194P + V205I + N261D S3L + S9R + G61E + A194P + N261D S9T + G61E + A194P + V205I + N261D S3F + S9R + G61E + A194P + N261D S9A + G61D + A194P + V205L + N261D S3V + S9R + G61E + A194P + N261D S9G + G61D + A194P + V205L + N261D S3Y + S9R + G61E + A194P + N261D S9M + G61D + A194P + V205L + N261D S3I + S9T + G61E + A194P + N261D S9R + G61D + A194P + V205L + N261D S3L + S9T + G61E + A194P + N261D S9T + G61D + A194P + V205L + N261D S3F + S9T + G61E + A194P + N261D S9A + G61E + A194P + V205L + N261D S3V + S9T + G61E + A194P + N261D S9G + G61E + A194P + V205L + N261D S3Y + S9T + G61E + A194P + N261D S9M + G61E + A194P + V205L + N261D S3I + S9A + A194P + V205I + N261D S9R + G61E + A194P + V205L + N261D S3L + S9A + A194P + V205I + N261D S9T + G61E + A194P + V205L + N261D S3F + S9A + A194P + V205I + N261D S9A + G61D + A194P + Q245H + N261D S3V + S9A + A194P + V205I + N261D S9G + G61D + A194P + Q245H + N261D S3Y + S9A + A194P + V205I + N261D S9M + G61D + A194P + Q245H + N261D S3I + S9G + A194P + V205I + N261D S9R + G61D + A194P + Q245H + N261D S3L + S9G + A194P + V205I + N261D S9T + G61D + A194P + Q245H + N261D S3F + S9G + A194P + V205I + N261D S9A + G61E + A194P + Q245H + N261D S3V + S9G + A194P + V205I + N261D S9G + G61E + A194P + Q245H + N261D S3Y + S9G + A194P + V205I + N261D S9M + G61E + A194P + Q245H + N261D S3I + S9M + A194P + V205I + N261D S9R + G61E + A194P + Q245H + N261D S3L + S9M + A194P + V205I + N261D S9T + G61E + A194P + Q245H + N261D S3F + S9M + A194P + V205I + N261D S9A + G61D + A194P + Q245K + N261D S3V + S9M + A194P + V205I + N261D S9G + G61D + A194P + Q245K + N261D S3Y + S9M + A194P + V205I + N261D S9M + G61D + A194P + Q245K + N261D S3I + S9R + A194P + V205I + N261D S9R + G61D + A194P + Q245K + N261D S3L + S9R + A194P + V205I + N261D S9T + G61D + A194P + Q245K + N261D S3F + S9R + A194P + V205I + N261D S9A + G61E + A194P + Q245K + N261D S3V + S9R + A194P + V205I + N261D S9G + G61E + A194P + Q245K + N261D S3Y + S9R + A194P + V205I + N261D S9M + G61E + A194P + Q245K + N261D S3I + S9T + A194P + V205I + N261D S9R + G61E + A194P + Q245K + N261D S3L + S9T + A194P + V205I + N261D S9T + G61E + A194P + Q245K + N261D S3F + S9T + A194P + V205I + N261D S9A + G61D + A194P + Q245R + N261D S3V + S9T + A194P + V205I + N261D S9G + G61D + A194P + Q245R + N261D S3Y + S9T + A194P + V205I + N261D S9M + G61D + A194P + Q245R + N261D S3I + S9A + A194P + V205L + N261D S9R + G61D + A194P + Q245R + N261D S3L + S9A + A194P + V205L + N261D S9T + G61D + A194P + Q245R + N261D S3F + S9A + A194P + V205L + N261D S9A + G61E + A194P + Q245R + N261D S3V + S9A + A194P + V205L + N261D S9G + G61E + A194P + Q245R + N261D S3Y + S9A + A194P + V205L + N261D S9M + G61E + A194P + Q245R + N261D S3I + S9G + A194P + V205L + N261D S9R + G61E + A194P + Q245R + N261D S3L + S9G + A194P + V205L + N261D S9T + G61E + A194P + Q245R + N261D S3F + S9G + A194P + V205L + N261D S9A + G61D + A194P + N261D + L262F S3V + S9G + A194P + V205L + N261D S9G + G61D + A194P + N261D + L262F S3Y + S9G + A194P + V205L + N261D S9M + G61D + A194P + N261D + L262F S3I + S9M + A194P + V205L + N261D S9R + G61D + A194P + N261D + L262F S3L + S9M + A194P + V205L + N261D S9T + G61D + A194P + N261D + L262F S3F + S9M + A194P + V205L + N261D S9A + G61E + A194P + N261D + L262F S3V + S9M + A194P + V205L + N261D S9G + G61E + A194P + N261D + L262F S3Y + S9M + A194P + V205L + N261D S9M + G61E + A194P + N261D + L262F S3I + S9R + A194P + V205L + N261D S9R + G61E + A194P + N261D + L262F S3L + S9R + A194P + V205L + N261D S9T + G61E + A194P + N261D + L262F S3F + S9R + A194P + V205L + N261D S9A + G61D + A194P + N261D + L262W S3V + S9R + A194P + V205L + N261D S9G + G61D + A194P + N261D + L262W S3Y + S9R + A194P + V205L + N261D S9M + G61D + A194P + N261D + L262W S3I + S9T + A194P + V205L + N261D S9R + G61D + A194P + N261D + L262W S3L + S9T + A194P + V205L + N261D S9T + G61D + A194P + N261D + L262W S3F + S9T + A194P + V205L + N261D S9A + G61E + A194P + N261D + L262W S3V + S9T + A194P + V205L + N261D S9G + G61E + A194P + N261D + L262W S3Y + S9T + A194P + V205L + N261D S9M + G61E + A194P + N261D + L262W S3I + S9A + A194P + Q245H + N261D S9R + G61E + A194P + N261D + L262W S3L + S9A + A194P + Q245H + N261D S9T + G61E + A194P + N261D + L262W S3F + S9A + A194P + Q245H + N261D S9A + G61D + A194P + N261D + L262Y S3V + S9A + A194P + Q245H + N261D S9G + G61D + A194P + N261D + L262Y S3Y + S9A + A194P + Q245H + N261D S9M + G61D + A194P + N261D + L262Y S3I + S9G + A194P + Q245H + N261D S9R + G61D + A194P + N261D + L262Y S3L + S9G + A194P + Q245H + N261D S9T + G61D + A194P + N261D + L262Y S3F + S9G + A194P + Q245H + N261D S9A + G61E + A194P + N261D + L262Y S3V + S9G + A194P + Q245H + N261D S9G + G61E + A194P + N261D + L262Y S3Y + S9G + A194P + Q245H + N261D S9M + G61E + A194P + N261D + L262Y S3I + S9M + A194P + Q245H + N261D S9R + G61E + A194P + N261D + L262Y S3L + S9M + A194P + Q245H + N261D S9T + G61E + A194P + N261D + L262Y S3F + S9M + A194P + Q245H + N261D S9A + A194P + V205I + Q245H + N261D S3V + S9M + A194P + Q245H + N261D S9G + A194P + V205I + Q245H + N261D S3Y + S9M + A194P + Q245H + N261D S9M + A194P + V205I + Q245H + N261D S3I + S9R + A194P + Q245H + N261D S9R + A194P + V205I + Q245H + N261D S3L + S9R + A194P + Q245H + N261D S9T + A194P + V205I + Q245H + N261D S3F + S9R + A194P + Q245H + N261D S9A + A194P + V205L + Q245H + N261D S3V + S9R + A194P + Q245H + N261D S9G + A194P + V205L + Q245H + N261D S3Y + S9R + A194P + Q245H + N261D S9M + A194P + V205L + Q245H + N261D S3I + S9T + A194P + Q245H + N261D S9R + A194P + V205L + Q245H + N261D S3L + S9T + A194P + Q245H + N261D S9T + A194P + V205L + Q245H + N261D S3F + S9T + A194P + Q245H + N261D S9A + A194P + V205I + Q245K + N261D S3V + S9T + A194P + Q245H + N261D S9G + A194P + V205I + Q245K + N261D S3Y + S9T + A194P + Q245H + N261D S9M + A194P + V205I + Q245K + N261D S3I + S9A + A194P + Q245K + N261D S9R + A194P + V205I + Q245K + N261D S3L + S9A + A194P + Q245K + N261D S9T + A194P + V205I + Q245K + N261D S3F + S9A + A194P + Q245K + N261D S9A + A194P + V205L + Q245K + N261D S3V + S9A + A194P + Q245K + N261D S9G + A194P + V205L + Q245K + N261D S3Y + S9A + A194P + Q245K + N261D S9M + A194P + V205L + Q245K + N261D S3I + S9G + A194P + Q245K + N261D S9R + A194P + V205L + Q245K + N261D S3L + S9G + A194P + Q245K + N261D S9T + A194P + V205L + Q245K + N261D S3F + S9G + A194P + Q245K + N261D S9A + A194P + V205I + Q245R + N261D S3V + S9G + A194P + Q245K + N261D S9G + A194P + V205I + Q245R + N261D S3Y + S9G + A194P + Q245K + N261D S9M + A194P + V205I + Q245R + N261D S3I + S9M + A194P + Q245K + N261D S9R + A194P + V205I + Q245R + N261D S3L + S9M + A194P + Q245K + N261D S9T + A194P + V205I + Q245R + N261D S3F + S9M + A194P + Q245K + N261D S9A + A194P + V205L + Q245R + N261D S3V + S9M + A194P + Q245K + N261D S9G + A194P + V205L + Q245R + N261D S3Y + S9M + A194P + Q245K + N261D S9M + A194P + V205L + Q245R + N261D S3I + S9R + A194P + Q245K + N261D S9R + A194P + V205L + Q245R + N261D S3L + S9R + A194P + Q245K + N261D S9T + A194P + V205L + Q245R + N261D S3F + S9R + A194P + Q245K + N261D S9A + A194P + V205I + N261 + L262F S3V + S9R + A194P + Q245K + N261D S9G + A194P + V205I + N261 + L262F S3Y + S9R + A194P + Q245K + N261D S9M + A194P + V205I + N261 + L262F S3I + S9T + A194P + Q245K + N261D S9R + A194P + V205I + N261 + L262F S3L + S9T + A194P + Q245K + N261D S9T + A194P + V205I + N261 + L262F S3F + S9T + A194P + Q245K + N261D S9A + A194P + V205L + N261 + L262F S3V + S9T + A194P + Q245K + N261D S9G + A194P + V205L + N261 + L262F S3Y + S9T + A194P + Q245K + N261D S9M + A194P + V205L + N261 + L262F S3I + S9A + A194P + Q245R + N261D S9R + A194P + V205L + N261 + L262F S3L + S9A + A194P + Q245R + N261D S9T + A194P + V205L + N261 + L262F S3F + S9A + A194P + Q245R + N261D S9A + A194P + V205I + N261 + L262W S3V + S9A + A194P + Q245R + N261D S9G + A194P + V205I + N261 + L262W S3Y + S9A + A194P + Q245R + N261D S9M + A194P + V205I + N261 + L262W S3I + S9G + A194P + Q245R + N261D S9R + A194P + V205I + N261 + L262W S3L + S9G + A194P + Q245R + N261D S9T + A194P + V205I + N261 + L262W S3F + S9G + A194P + Q245R + N261D S9A + A194P + V205L + N261 + L262W S3V + S9G + A194P + Q245R + N261D S9G + A194P + V205L + N261 + L262W S3Y + S9G + A194P + Q245R + N261D S9M + A194P + V205L + N261 + L262W S3I + S9M + A194P + Q245R + N261D S9R + A194P + V205L + N261 + L262W S3L + S9M + A194P + Q245R + N261D S9T + A194P + V205L + N261 + L262W S3F + S9M + A194P + Q245R + N261D S9A + A194P + V205I + N261 + L262Y S3V + S9M + A194P + Q245R + N261D S9G + A194P + V205I + N261 + L262Y S3Y + S9M + A194P + Q245R + N261D S9M + A194P + V205I + N261 + L262Y S3I + S9R + A194P + Q245R + N261D S9R + A194P + V205I + N261 + L262Y S3L + S9R + A194P + Q245R + N261D S9T + A194P + V205I + N261 + L262Y S3F + S9R + A194P + Q245R + N261D S9A + A194P + V205L + N261 + L262Y S3V + S9R + A194P + Q245R + N261D S9G + A194P + V205L + N261 + L262Y S3Y + S9R + A194P + Q245R + N261D S9M + A194P + V205L + N261 + L262Y S3I + S9T + A194P + Q245R + N261D S9R + A194P + V205L + N261 + L262Y S3L + S9T + A194P + Q245R + N261D S9T + A194P + V205L + N261 + L262Y S3F + S9T + A194P + Q245R + N261D S9A + A194P + Q245H + N261 + L262F S3V + S9T + A194P + Q245R + N261D S9G + A194P + Q245H + N261 + L262F S3Y + S9T + A194P + Q245R + N261D S9M + A194P + Q245H + N261 + L262F S3I + S9A + A194P + N261D + L262F S9R + A194P + Q245H + N261 + L262F S3L + S9A + A194P + N261D + L262F S9T + A194P + Q245H + N261 + L262F S3F + S9A + A194P + N261D + L262F S9A + A194P + Q245R + N261 + L262F S3V + S9A + A194P + N261D + L262F S9G + A194P + Q245R + N261 + L262F S3Y + S9A + A194P + N261D + L262F S9M + A194P + Q245R + N261 + L262F S3I + S9G + A194P + N261D + L262F S9R + A194P + Q245R + N261 + L262F S3L + S9G + A194P + N261D + L262F S9T + A194P + Q245R + N261 + L262F S3F + S9G + A194P + N261D + L262F S9A + A194P + Q245T + N261 + L262F S3V + S9G + A194P + N261D + L262F S9G + A194P + Q245T + N261 + L262F S3Y + S9G + A194P + N261D + L262F S9M + A194P + Q245T + N261 + L262F S3I + S9M + A194P + N261D + L262F S9R + A194P + Q245T + N261 + L262F S3L + S9M + A194P + N261D + L262F S9T + A194P + Q245T + N261 + L262F S3F + S9M + A194P + N261D + L262F S9A + A194P + Q245H + N261 + L262W S3V + S9M + A194P + N261D + L262F S9G + A194P + Q245H + N261 + L262W S3Y + S9M + A194P + N261D + L262F S9M + A194P + Q245H + N261 + L262W S3I + S9R + A194P + N261D + L262F S9R + A194P + Q245H + N261 + L262W S3L + S9R + A194P + N261D + L262F S9T + A194P + Q245H + N261 + L262W S3F + S9R + A194P + N261D + L262F S9A + A194P + Q245R + N261 + L262W S3V + S9R + A194P + N261D + L262F S9G + A194P + Q245R + N261 + L262W S3Y + S9R + A194P + N261D + L262F S9M + A194P + Q245R + N261 + L262W S3I + S9T + A194P + N261D + L262F S9R + A194P + Q245R + N261 + L262W S3L + S9T + A194P + N261D + L262F S9T + A194P + Q245R + N261 + L262W S3F + S9T + A194P + N261D + L262F S9A + A194P + Q245T + N261 + L262W S3V + S9T + A194P + N261D + L262F S9G + A194P + Q245T + N261 + L262W S3Y + S9T + A194P + N261D + L262F S9M + A194P + Q245T + N261 + L262W S3I + S9A + A194P + N261D + L262W S9R + A194P + Q245T + N261 + L262W S3L + S9A + A194P + N261D + L262W S9T + A194P + Q245T + N261 + L262W S3F + S9A + A194P + N261D + L262W S9A + A194P + Q245H + N261 + L262Y S3V + S9A + A194P + N261D + L262W S9G + A194P + Q245H + N261 + L262Y S3Y + S9A + A194P + N261D + L262W S9M + A194P + Q245H + N261 + L262Y S3I + S9G + A194P + N261D + L262W S9R + A194P + Q245H + N261 + L262Y S3L + S9G + A194P + N261D + L262W S9T + A194P + Q245H + N261 + L262Y S3F + S9G + A194P + N261D + L262W S9A + A194P + Q245R + N261 + L262Y S3V + S9G + A194P + N261D + L262W S9G + A194P + Q245R + N261 + L262Y S3Y + S9G + A194P + N261D + L262W S9M + A194P + Q245R + N261 + L262Y S3I + S9M + A194P + N261D + L262W S9R + A194P + Q245R + N261 + L262Y S3L + S9M + A194P + N261D + L262W S9T + A194P + Q245R + N261 + L262Y S3F + S9M + A194P + N261D + L262W S9A + A194P + Q245T + N261 + L262Y S3V + S9M + A194P + N261D + L262W S9G + A194P + Q245T + N261 + L262Y S3Y + S9M + A194P + N261D + L262W S9M + A194P + Q245T + N261 + L262Y S3I + S9R + A194P + N261D + L262W S9R + A194P + Q245T + N261 + L262Y S3L + S9R + A194P + N261D + L262W S9T + A194P + Q245T + N261 + L262Y S3F + S9R + A194P + N261D + L262W N43D + G61D + A194P + V205I + N261D S3V + S9R + A194P + N261D + L262W N43E + G61D + A194P + V205I + N261D S3Y + S9R + A194P + N261D + L262W N43D + G61E + A194P + V205I + N261D S3I + S9T + A194P + N261D + L262W N43E + G61E + A194P + V205I + N261D S3L + S9T + A194P + N261D + L262W N43D + G61D + A194P + V205L + N261D S3F + S9T + A194P + N261D + L262W N43E + G61D + A194P + V205L + N261D S3V + S9T + A194P + N261D + L262W N43D + G61E + A194P + V205L + N261D S3Y + S9T + A194P + N261D + L262W N43E + G61E + A194P + V205L + N261D S3I + S9A + A194P + N261D + L262Y N43D + G61D + A194P + Q245H + N261D S3L + S9A + A194P + N261D + L262Y N43E + G61D + A194P + Q245H + N261D S3F + S9A + A194P + N261D + L262Y N43D + G61E + A194P + Q245H + N261D S3V + S9A + A194P + N261D + L262Y N43E + G61E + A194P + Q245H + N261D S3Y + S9A + A194P + N261D + L262Y N43D + G61D + A194P + Q245K + N261D S3I + S9G + A194P + N261D + L262Y N43E + G61D + A194P + Q245K + N261D S3L + S9G + A194P + N261D + L262Y N43D + G61E + A194P + Q245K + N261D S3F + S9G + A194P + N261D + L262Y N43E + G61E + A194P + Q245K + N261D S3V + S9G + A194P + N261D + L262Y N43D + G61D + A194P + Q245R + N261D S3Y + S9G + A194P + N261D + L262Y N43E + G61D + A194P + Q245R + N261D S3I + S9M + A194P + N261D + L262Y N43D + G61E + A194P + Q245R + N261D S3L + S9M + A194P + N261D + L262Y N43E + G61E + A194P + Q245R + N261D S3F + S9M + A194P + N261D + L262Y N43D + G61D + A194P + N261D + L262F S3V + S9M + A194P + N261D + L262Y N43E + G61D + A194P + N261D + L262F S3Y + S9M + A194P + N261D + L262Y N43D + G61E + A194P + N261D + L262F S3I + S9R + A194P + N261D + L262Y N43E + G61E + A194P + N261D + L262F S3L + S9R + A194P + N261D + L262Y N43D + G61D + A194P + N261D + L262W S3F + S9R + A194P + N261D + L262Y N43E + G61D + A194P + N261D + L262W S3V + S9R + A194P + N261D + L262Y N43D + G61E + A194P + N261D + L262W S3Y + S9R + A194P + N261D + L262Y N43E + G61E + A194P + N261D + L262W S3I + S9T + A194P + N261D + L262Y N43D + G61D + A194P + N261D + L262Y S3L + S9T + A194P + N261D + L262Y N43E + G61D + A194P + N261D + L262Y S3F + S9T + A194P + N261D + L262Y N43D + G61E + A194P + N261D + L262Y S3V + S9T + A194P + N261D + L262Y N43E + G61E + A194P + N261D + L262Y S3Y + S9T + A194P + N261D + L262Y N43D + A194P + V205I + Q245H + N261D S3I + N43D + G61D + A194P + N261D N43E + A194P + V205I + Q245H + N261D S3L + N43D + G61D + A194P + N261D N43D + A194P + V205L + Q245H + N261D S3F + N43D + G61D + A194P + N261D N43E + A194P + V205L + Q245H + N261D S3V + N43D + G61D + A194P + N261D N43D + A194P + V205I + Q245K + N261D S3Y + N43D + G61D + A194P + N261D N43E + A194P + V205I + Q245K + N261D S3I + N43E + G61D + A194P + N261D N43D + A194P + V205L + Q245K + N261D S3L + N43E + G61D + A194P + N261D N43E + A194P + V205L + Q245K + N261D S3F + N43E + G61D + A194P + N261D N43D + A194P + V205I + Q245R + N261D S3V + N43E + G61D + A194P + N261D N43E + A194P + V205I + Q245R + N261D S3Y + N43E + G61D + A194P + N261D N43D + A194P + V205L + Q245R + N261D S3I + N43D + G61E + A194P + N261D N43E + A194P + V205L + Q245R + N261D S3L + N43D + G61E + A194P + N261D N43D + A194P + V205I + N261D + L262F S3F + N43D + G61E + A194P + N261D N43E + A194P + V205I + N261D + L262F S3V + N43D + G61E + A194P + N261D N43D + A194P + V205L + N261D + L262F S3Y + N43D + G61E + A194P + N261D N43E + A194P + V205L + N261D + L262F S3I + N43E + G61E + A194P + N261D N43D + A194P + V205I + N261D + L262W S3L + N43E + G61E + A194P + N261D N43E + A194P + V205I + N261D + L262W S3F + N43E + G61E + A194P + N261D N43D + A194P + V205L + N261D + L262W S3V + N43E + G61E + A194P + N261D N43E + A194P + V205L + N261D + L262W S3Y + N43E + G61E + A194P + N261D N43D + A194P + V205I + N261D + L262Y S3I + N43D + A194P + V205I + N261D N43E + A194P + V205I + N261D + L262Y S3L + N43D + A194P + V205I + N261D N43D + A194P + V205L + N261D + L262Y S3F + N43D + A194P + V205I + N261D N43E + A194P + V205L + N261D + L262Y S3V + N43D + A194P + V205I + N261D N43D + A194P + Q245H + N261D + L262F S3Y + N43D + A194P + V205I + N261D N43E + A194P + Q245H + N261D + L262F S3I + N43E + A194P + V205I + N261D N43D + A194P + Q245R + N261D + L262F S3L + N43E + A194P + V205I + N261D N43E + A194P + Q245R + N261D + L262F S3F + N43E + A194P + V205I + N261D N43D + A194P + Q245T + N261D + L262F S3V + N43E + A194P + V205I + N261D N43E + A194P + Q245T + N261D + L262F S3Y + N43E + A194P + V205I + N261D N43D + A194P + Q245H + N261D + L262W S3I + N43D + A194P + V205L + N261D N43E + A194P + Q245H + N261D + L262W S3L + N43D + A194P + V205L + N261D N43D + A194P + Q245R + N261D + L262W S3F + N43D + A194P + V205L + N261D N43E + A194P + Q245R + N261D + L262W S3V + N43D + A194P + V205L + N261D N43D + A194P + Q245T + N261D + L262W S3Y + N43D + A194P + V205L + N261D N43E + A194P + Q245T + N261D + L262W S3I + N43E + A194P + V205L + N261D N43D + A194P + Q245H + N261D + L262Y S3L + N43E + A194P + V205L + N261D N43E + A194P + Q245H + N261D + L262Y S3F + N43E + A194P + V205L + N261D N43D + A194P + Q245R + N261D + L262Y S3V + N43E + A194P + V205L + N261D N43E + A194P + Q245R + N261D + L262Y S3Y + N43E + A194P + V205L + N261D N43D + A194P + Q245T + N261D + L262Y S3I + N43D + A194P + Q245H + N261D N43E + A194P + Q245T + N261D + L262Y S3L + N43D + A194P + Q245H + N261D G61D + A194P + V205I + Q245H + N261D S3F + N43D + A194P + Q245H + N261D G61E + A194P + V205I + Q245H + N261D S3V + N43D + A194P + Q245H + N261D G61D + A194P + V205L + Q245H + N261D S3Y + N43D + A194P + Q245H + N261D G61E + A194P + V205L + Q245H + N261D S3I + N43E + A194P + Q245H + N261D G61D + A194P + V205I + Q245K + N261D S3L + N43E + A194P + Q245H + N261D G61E + A194P + V205I + Q245K + N261D S3F + N43E + A194P + Q245H + N261D G61D + A194P + V205L + Q245K + N261D S3V + N43E + A194P + Q245H + N261D G61E + A194P + V205L + Q245K + N261D S3Y + N43E + A194P + Q245H + N261D G61D + A194P + V205I + Q245R + N261D S3I + N43D + A194P + Q245K + N261D G61E + A194P + V205I + Q245R + N261D S3L + N43D + A194P + Q245K + N261D G61D + A194P + V205L + Q245R + N261D S3F + N43D + A194P + Q245K + N261D G61E + A194P + V205L + Q245R + N261D S3V + N43D + A194P + Q245K + N261D G61D + A194P + V205I + N261D + L262F S3Y + N43D + A194P + Q245K + N261D G61E + A194P + V205I + N261D + L262F S3I + N43E + A194P + Q245K + N261D G61D + A194P + V205L + N261D + L262F S3L + N43E + A194P + Q245K + N261D G61E + A194P + V205L + N261D + L262F S3F + N43E + A194P + Q245K + N261D G61D + A194P + V205I + N261D + L262W S3V + N43E + A194P + Q245K + N261D G61E + A194P + V205I + N261D + L262W S3Y + N43E + A194P + Q245K + N261D G61D + A194P + V205L + N261D + L262W S3I + N43D + A194P + Q245R + N261D G61E + A194P + V205L + N261D + L262W S3L + N43D + A194P + Q245R + N261D G61D + A194P + V205I + N261D + L262Y S3F + N43D + A194P + Q245R + N261D G61E + A194P + V205I + N261D + L262Y S3V + N43D + A194P + Q245R + N261D G61D + A194P + V205L + N261D + L262Y S3Y + N43D + A194P + Q245R + N261D G61E + A194P + V205L + N261D + L262Y S3I + N43E + A194P + Q245R + N261D G61D + A194P + Q245H + N261D + L262F S3L + N43E + A194P + Q245R + N261D G61E + A194P + Q245H + N261D + L262F S3F + N43E + A194P + Q245R + N261D G61D + A194P + Q245R + N261D + L262F S3V + N43E + A194P + Q245R + N261D G61E + A194P + Q245R + N261D + L262F S3Y + N43E + A194P + Q245R + N261D G61D + A194P + Q245T + N261D + L262F S3I + N43D + A194P + N261 + L262F G61E + A194P + Q245T + N261D + L262F S3L + N43D + A194P + N261 + L262F G61D + A194P + Q245H + N261D + L262W S3F + N43D + A194P + N261 + L262F G61E + A194P + Q245H + N261D + L262W S3V + N43D + A194P + N261 + L262F G61D + A194P + Q245R + N261D + L262W S3Y + N43D + A194P + N261 + L262F G61E + A194P + Q245R + N261D + L262W S3I + N43E + A194P + N261 + L262F G61D + A194P + Q245T + N261D + L262W S3L + N43E + A194P + N261 + L262F G61E + A194P + Q245T + N261D + L262W S3F + N43E + A194P + N261 + L262F G61D + A194P + Q245H + N261D + L262Y S3V + N43E + A194P + N261 + L262F G61E + A194P + Q245H + N261D + L262Y S3Y + N43E + A194P + N261 + L262F G61D + A194P + Q245R + N261D + L262Y S3I + N43D + A194P + N261 + L262W G61E + A194P + Q245R + N261D + L262Y S3L + N43D + A194P + N261 + L262W G61D + A194P + Q245T + N261D + L262Y S3F + N43D + A194P + N261 + L262W G61E + A194P + Q245T + N261D + L262Y S3V + N43D + A194P + N261 + L262W A194P + V205I + Q245H + N261D + L262F S3Y + N43D + A194P + N261 + L262W A194P + V205L + Q245H + N261D + L262F S3I + N43E + A194P + N261 + L262W A194P + V205I + Q245R + N261D + L262F S3L + N43E + A194P + N261 + L262W A194P + V205L + Q245R + N261D + L262F S3F + N43E + A194P + N261 + L262W A194P + V205I + Q245T + N261D + L262F S3V + N43E + A194P + N261 + L262W A194P + V205L + Q245T + N261D + L262F S3Y + N43E + A194P + N261 + L262W A194P + V205I + Q245H + N261D + L262W S3I + N43D + A194P + N261 + L262Y A194P + V205L + Q245H + N261D + L262W S3L + N43D + A194P + N261 + L262Y A194P + V205I + Q245R + N261D + L262W S3F + N43D + A194P + N261 + L262Y A194P + V205L + Q245R + N261D + L262W S3V + N43D + A194P + N261 + L262Y A194P + V205I + Q245T + N261D + L262W S3Y + N43D + A194P + N261 + L262Y A194P + V205L + Q245T + N261D + L262W S3I + N43E + A194P + N261 + L262Y A194P + V205I + Q245H + N261D + L262Y S3L + N43E + A194P + N261 + L262Y A194P + V205L + Q245H + N261D + L262Y S3F + N43E + A194P + N261 + L262Y A194P + V205I + Q245R + N261D + L262Y S3V + N43E + A194P + N261 + L262Y A194P + V205L + Q245R + N261D + L262Y S3Y + N43E + A194P + N261 + L262Y A194P + V205I + Q245T + N261D + L262Y S3I + G61D + A194P + V205I + N261D A194P + V205L + Q245T + N261D + L262Y

The subtilase variants may further comprise a substitution at one or more positions (e.g. several) selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270, preferably positions 15, 63, 68 and/or 217 (numbering according to SEQ ID NO: 2). It will be clear to the skilled artisan that if a position has already been altered once, then it will not be altered a second time.). In a more preferred embodiment, the subtilase variant further comprises one or more substitutions selected from the group consisting of 4I, 9{H, K, R}, 12{D, E}, 14T, 15{G, M, S, T}, 40{D, E}, 63G, 68{A, G, 1, L, M, S, T}, 79T, 86H, 88V, 92S, 98T, 101L, 146S, 188{A, G, M, T}, 217L, 245R and/or 270{G, M, S, T} (numbering according to SEQ ID NO: 2). In an even more preferred embodiment, the subtilase variant further comprises one or more substitutions selected from the group consisting of V4I, S9{R, Q12E, P14T, A15T, P40E, S63G, V68A, I79T, P86H, A88V, A92S, A98T, S101L, G146S, S188T, Y217L, Q245R, and/or A270G and/or a deletion in position S132* in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2. In a particularly preferred embodiment of the invention the variants in table 2 are combined with A15T and/or V68A. Thus a preferred embodiment of the invention concerns any variants of table 2+A15T, any variants of table 2+V68A or any variants of table 2+A15T+V68A and particularly the specific variants of table 3.

TABLE 3 S3V + S9R + G61E + V68A + A194P + N261D N43D + A15T + G61E + A194P + Q245R + N261D S3V + S9R + A15T + G61E + A194P + N261D N43D + G61E + V68A + A194P + Q245R + N261D S3V + S9R + A15T + G61E + V68A + A194P + N43D + A15T + G61E + V68A + A194P + Q245R + N261D N261D S3V + A15T + N43D + A194P + Q245R + N261D S9R + A15T + G61E + A194P + V205I + N261D S3V + N43D + V68A + A194P + Q245R + N261D S9R + G61E + V68A + A194P + V205I + N261D S3V + A15T + N43D + V68A + A194P + Q245R + S9R + A15T + G61E + V68A + A194P + V205I + N261D N261D S3V + S9R + A15T + N43D + A194P + Q245R + S9R + A15T + A194P + Q245R + N261D N261D S3V + S9R + N43D + V68A + A194P + Q245R + S9R + V68A + A194P + Q245R + N261D N261D S3V + S9R + A15T + N43D + V68A + A194P + S9R + A15T + V68A + A194P + Q245R + N261D Q245R + N261D S3V + A15T + A194P + Q245R + N261D S9R + A15T + G61E + A194P + V205I + Q245R + N261D S3V + V68A + A194P + Q245R + N261D S9R + G61E + V68A + A194P + V205I + Q245R + N261D S3V + A15T + V68A + A194P + Q245R + N261D S9R + A15T + G61E + V68A + A194P + V205I + Q245R + N261D S3V + S9R + G61E + A194P + V205I + Q245R + S9R + G61E + A194P + V205I + N261D N261D S3V + S9R + A15T + G61E + A194P + V205I + A15T + N43E + G61D + A194P + Q245R + N261D Q245R + N261D S3V + S9R + G61E + V68A + A194P + V205I + N43E + G61D + V68A + A194P + Q245R + N261D Q245R + N261D S3V + S9R + A15T + G61E + V68A + A194P + A15T + N43E + G61D + V68A + A194P + Q245R + V205I + Q245R + N261D N261D S9R + A15T + G61E + A194P + V205I + Q245R A15T + G61E + A194P + Q245T + N261D + L262Y S9R + G61E + V68A + A194P + V205I + Q245R G61E + V68A + A194P + Q245T + N261D + L262Y S9R + A15T + G61E + V68A + A194P + V205I + A15T + G61E + V68A + A194P + Q245T + N261D + Q245R L262Y S9R + A15T + A194P + Q245R + N261D G61E + A15T + A194P + Q245R + N261D + L262Y S9R + V68A + A194P + Q245R + N261D G61E + V68A + A194P + Q245R + N261D + L262Y S9R + A15T + V68A + A194P + Q245R + N261D G61E + A15T + V68A + A194P + Q245R + N261D + L262Y S9R + A15T + A194P + Q245R + N261D + A15T + A194P + V205I + Q245T + N261D + L262Y L262Y S9R + V68A + A194P + Q245R + N261D + V68A + A194P + V205I + Q245T + N261D + L262Y L262Y S9R + A15T + V68A + A194P + Q245R + N261D + A15T + V68A + A194P + V205I + Q245T + N261D + L262Y L262Y

The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. For Savinase (SEQ ID NO: 4) the catalytic triad comprising the amino acids S221, H64, and D32 (BPN′ numbering) is essential for protease activity of the enzyme.

The subtilase variants may consist of 150 to 350, e.g., 175 to 330, 200 to 310, 220 to 300, 240 to 290, 260 to 280 or 269, 270, 271, 272, 273, 274 or 275 amino acids.

In one embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4. In a preferred embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of the parent subtilase or to the mature polypeptide of SEQ ID NO: 4.

In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the parent enzyme wherein in wash stability is measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein. In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of SEQ ID NO: 4 wherein in wash stability is measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the parent enzyme wherein in wash stability is measured using the ‘in wash stability assay’ and wash performance is measured using the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash as described in the Materials and Methods section herein. In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of SEQ ID NO: 4 wherein in wash stability is measured using the ‘In Wash Stability Assay’ and wash performance is measured using the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash as described in the Materials and Methods section herein.

Parent Protease

Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).

Serine Proteases

A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 “Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).

The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41:711-753).

Subtilases

A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al. (1991), Protein Eng. 4:719-737 and Siezen et al. (1997), Protein Science 6:501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).

Subtilisins

A subgroup of the subtilases is the subtilisins which are serine proteases from the family S8, in particular from the subfamily S8A, as defined by the MEROPS database (http://merops.sanger.ac.uk/cgi-bin/famsum?family=S8).

BPN′ and Savinase have the MEROPS numbers S08.034 and S08.003, respectively.

Parent Subtilase

The term “parent subtilase” describes a subtilase defined according to Siezen et al. (1997), Protein Science 6:501-523. For further details see description of “Subtilases” above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications (such as replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertion(s)) have been made while retaining the characteristic of a subtilase. Furthermore, a parent subtilase may be a subtilase which has been prepared by the DNA shuffling technique, such as described by J. E. Ness et al. (1999) Nature Biotechnology, 17:893-896.

Alternatively, the term “parent subtilase” may be termed “wild type subtilase”. The parent subtilase is preferably of the subtilisin subgroups. One subgroup of the subtilases, I-S1 or “true” subtilisins, comprises the “classical” subtilisins, such as subtilisin 168 (BSS168), subtilisin BPN′, subtilisin Carlsberg (ALCALASE®, Novozymes A/S), and subtilisin DY (BSSDY).

A further subgroup of the subtilases, I-S2 or high alkaline subtilisins, is recognized by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL®, Genencor International Inc.), subtilisin 309 (SAVINASE®, Novozymes A/S), subtilisin 147 (BLS147) (ESPERASE®, Novozymes A/S), and alkaline elastase YaB (BSEYAB). BPN′ is subtilisin BPN′ from B. amyloliquefaciens BPN′ has the amino acid sequence SEQ ID NO 2.

For reference, table 3 below gives a list of some acronyms for various subtilases mentioned herein. For further acronyms, see Siezen et al. (1991 and 1997).

TABLE 3 Acronyms of various subtilases Organism Enzyme Acronym Bacillus subtilis 168 subtilisin I168, apr BSS168 Bacillus amyloliquefaciens subtilisin BPN′ (NOVO) BASBPN Bacillus subtilis DY subtilisin DY BSSDY Bacillus licheniformis subtilisin Carlsberg BLSCAR Bacillus lentus subtilisin 309 BLSAVI Bacillus lentus subtilisin 147 BLS147 Bacillus alcalophilus PB92 subtilisin PB92 BAPB92 Bacillus YaB alkaline elastase YaB BYSYAB Bacillus sp. NKS-21 subtilisin ALP I BSAPRQ Bacillus sp. G-825-6 subtilisin Sendai BSAPRS Thermoactinomyces vulgaris Thermitase TVTHER

Homologous Subtilase Sequences

The homology between two amino acid sequences is in this context described by the parameter “identity” for purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as described above. The output from the routine is besides the amino acid alignment the calculation of the “Percent Identity” between the two sequences.

Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases, which can be modified according to the invention.

Substantially homologous parent subtilisin variants may have one or more (several) amino acid substitutions, deletions and/or insertions, in the present context the term “one or more” is used interchangeably with the term “several”. These changes are preferably of a minor nature, that is conservative amino acid substitutions as described above and other substitutions that do not significantly affect the three-dimensional folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tract, or protein (Nilsson et al. (1985) EMBO J. 4: 1075; Nilsson et al. (1991) Methods Enzymol. 198:3. See, also, in general, Ford et al. (1991) Protein Expression and Purification 2: 95-107.

Although the changes described above preferably are of a minor nature, such changes may also be of a substantive nature such as fusion of larger polypeptides of up to 300 amino acids or more both as amino- or carboxyl-terminal extensions.

The parent protease may be (a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the parent comprises or consists of amino acids 1 to 275 of SEQ ID NO: 2. In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another aspect, the nucleotide acid probe is a 80 to 1140 nucleotides long fragment of SEQ ID NO: 1 e.g. 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 nucleotides long. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2 respectively.

In another embodiment, the parent is encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The parent protease may be (a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 4; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3, or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3.

In an aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 4.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 4. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 4. In another aspect, the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO: 4. In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 4.

In another aspect, the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3, or (ii) the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 3 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 4 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 3 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 3; (ii) the mature polypeptide coding sequence of SEQ ID NO: 3; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 3. In another aspect, the nucleotide acid probe is a 80 to 1140 nucleotides long fragment of SEQ ID NO: 3 e.g. 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 nucleotides long. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 4; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 3 or a sequence encoding the mature polypeptide of SEQ ID NO: 4 respectively.

In another embodiment, the parent is encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces protease, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis protease In one aspect, the parent is a Bacillus amyloliquefaciens protease, e.g., the protease of SEQ ID NO: 2 or the mature polypeptide thereof.

In another aspect, the parent is a Bacillus lentus protease, e.g., the protease of SEQ ID NO: 4 or the mature polypeptide thereof.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a subtilase variant having protease activity, comprising: (a) introducing into a parent subtilase the double substitution 194P+261D and optionally one or more alterations from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2, and (b) recovering the variant.

In an embodiment, the invention relates to a method for obtaining a subtilase variant having protease activity, comprising:

-   -   a) introducing into a parent subtilase the double substitution         194P+261D and one or more alterations from the group consisting         of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T},         40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E},         72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E},         120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E},         183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D,         E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D,         E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position         corresponds to the position of the mature polypeptide of SEQ ID         NO: 2 and wherein the subtilase variant is selected from the         list consisting of:         -   1) a polypeptide that has at least 60%, e.g., at least 65%,             at least 70%, at least 75%, at least 80%, at least 85%, at             least 90%, at least 95%, at least 96%, at least 97%, at             least 98% but less than 100% sequence identity to the mature             polypeptide of the parent subtilase;         -   2) a polypeptide that is encoded by a polynucleotide that             hybridizes under low stringency conditions, medium             stringency conditions, medium-high stringency conditions,             high stringency conditions, or very high stringency             conditions with             -   (i) the mature polypeptide coding sequence of the parent                 subtilase or             -   (ii) the full-length complement of (i); and         -   3) a polypeptide that is encoded by a polynucleotide having             at least 60%, e.g., at least 65%, at least 70%, at least             75%, at least 80%, at least 85%, at least 90%, at least 95%,             at least 96%, at least 97%, at least 98% but less than 100%             sequence identity to the mature polypeptide coding sequence             of the parent subtilase; and     -   b) recovering the variant.

The subtilase variants may further comprise a substitution, deletion and/or insertion at one or more positions (e.g. several) selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270, preferably positions 15, 63, 68 and/or 217 (numbering according to SEQ ID NO: 2). It will be clear to the skilled artisan that if a position has already been altered once, then it will not be altered a second time. In a preferred embodiment, the alteration at one or more positions (e.g. several) that is selected from the group consisting of 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 146, 188, 217, 245, 255 and 270 is a substitution or for position 132 is a deletion (numbering according to SEQ ID NO: 2).

In another embodiment, the invention relates to a method for obtaining a subtilase variant having protease activity, comprising:

-   -   a) introducing into mature polypeptide of SEQ ID NO: 4 the         double substitution A194P+N261D and one or more alterations from         the group consisting of S3{I, L, P, V, Y}, S9{A, G, M, T},         N18{A, G, M, S, T}, P40{D, E}, N43{D, E}, R45{D, E}, T58{F, Y},         Q59{D, E}, G61{D, E}, 172{L, V}, N76{D, E}, S99{A, E, G, M, T},         V104{F, Y}, S105{D, E}, H120{D, E, I, L, V}, S130*, A133{D, E},         5141{F, Y}, Q182{D, E}, N183{D, E}, N184{D, E}, V205{I, L},         Q206{D, E}, S212E, N218{D, E}, T224{A, G, M, S}, P225{A, G, M,         S, T}, A228{G, M, S, T}, Q236{D, E}, Q245{H, K}, 5259{D, E} and         L262{F, W, Y} wherein the position corresponds to the position         of the mature polypeptide of SEQ ID NO: 2 and wherein the         subtilase variant is selected from the list consisting of:         -   1) a polypeptide that has at least 60%, e.g., at least 65%,             at least 70%, at least 75%, at least 80%, at least 85%, at             least 90%, at least 95%, at least 96%, at least 97%, at             least 98% but less than 100% sequence identity to the mature             polypeptide of SEQ ID NO: 4;         -   2) a polypeptide that is encoded by a polynucleotide that             hybridizes under low stringency conditions, medium             stringency conditions, medium-high stringency conditions,             high stringency conditions, or very high stringency             conditions with             -   (i) the mature polypeptide coding sequence of SEQ ID NO:                 3 or             -   (ii) the full-length complement of (i); and         -   3) a polypeptide that is encoded by a polynucleotide having             at least 60%, e.g., at least 65%, at least 70%, at least             75%, at least 80%, at least 85%, at least 90%, at least 95%,             at least 96%, at least 97%, at least 98% but less than 100%             sequence identity to the mature polypeptide coding sequence             of SEQ ID NO: 3; and     -   b) recovering the variant.

A preferred embodiment, the invention relates to a method for obtaining a subtilase variant having protease activity, comprising:

a) introducing into a parent subtilase the double substitution 194P+261D and one or more alterations from the group consisting of 3V, 3Y, 9M, 18S, 18T, 18D, 40E, 43D, 43E, 45E, 58Y, 59E, 61D, 72V, 76D, 99D, 104Y, 1054D, 120I, 120D, 130*, 133E, 141F, 182E, 183D, 184D, 205I, 212D, 218D, 228S, 236E and 262Y wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 and wherein the subtilase variant is selected from the list consisting of:

-   -   1) a polypeptide that has at least 60%, e.g., at least 65%, at         least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 95%, at least 96%, at least 97%, at least 98% but         less than 100% sequence identity to the mature polypeptide of         the parent subtilase;     -   2) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with         -   (i) the mature polypeptide coding sequence of the parent             subtilase or         -   (ii) the full-length complement of (i); and     -   3) a polypeptide that is encoded by a polynucleotide having at         least 60%, e.g., at least 65%, at least 70%, at least 75%, at         least 80%, at least 85%, at least 90%, at least 95%, at least         96%, at least 97%, at least 98% but less than 100% sequence         identity to the mature polypeptide coding sequence of the parent         subtilase; and

b) recovering the variant.

The subtilase variants may further comprise a substitution at one or more positions (e.g. several) selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270, preferably positions 15, 63, 68 and/or 217 (numbering according to SEQ ID NO: 2). It will be clear to the skilled artisan that if a position has already been altered once, then it will not be altered a second time. In one embodiment, position 132 is deleted (numbering according to SEQ ID NO: 2). In a preferred embodiment, the subtilase variant further comprises one or more substitutions selected from the group consisting of 4I, 9{H, K, R}, 12{D, E}, 14T, 15{G, M, S, T}, 40{D, E}, 63G, 68{A, G, I, L, M, S, T}, 79T, 86H, 88V, 92S, 98T, 101L, 146S, 188{A, G, M, T}, 217L, 245R and/or 270{G, M, S, T} (numbering according to SEQ ID NO: 2). In an even more preferred embodiment, the subtilase variant further comprises one or more substitutions selected from the group consisting of V4I, S9{R, Q12E, P14T, A15T, P40E, S63G, V68A, I79T, P86H, A88V, A92S, A98T, S101L, G146S, S188T, Y217L, Q245R, and/or A270G and/or a deletion in position S132* in the mature polypeptide of SEQ ID NO: 4, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO: 2.

In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the parent enzyme wherein in wash stability is measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein. In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, compared to the mature polypeptide of SEQ ID NO: 4 wherein in wash stability is measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the parent enzyme wherein in wash stability is measured using the ‘in wash stability assay’ and wash performance is measured using the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash as described in the Materials and Methods section herein. In an embodiment, the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance compared to the mature polypeptide of SEQ ID NO: 4 wherein in wash stability is measured using the ‘In Wash Stability Assay’ and wash performance is measured using the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash as described in the Materials and Methods section herein.

The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which is recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.

Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of the variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

Methods of Production

The present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the variant to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that are specific for the variants with protease activity. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The variant may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing the variant is used as a source of the variant.

Compositions

In one certain aspect, the variants according to the invention have improved wash performance compared to the parent enzyme or compared to a protease having the identical amino acid sequence of said variant but not having the alterations at one or more of said specified positions or compared to the mature polypeptide of SEQ ID NO: 4, wherein wash performance is measured using the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash as described in the Materials and Methods section herein.

In another certain aspect, the variants according to the invention have improved stability, preferably improved storage stability, compared to the parent enzyme or compared to a protease having the identical amino acid sequence of said variant but not having the alterations at one or more of said specified positions or compared to the mature polypeptide of SEQ ID NO: 4, wherein storage stability is measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

Thus, in a preferred embodiment the composition is a detergent composition, and one aspect of the invention relates to the use of a detergent composition comprising a variant according to the invention in a cleaning process such as laundry or hard surface cleaning.

The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.

Enzyme of the Present Invention

In one embodiment of the present invention, the a polypeptide of the present invention may be added to a detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.

A composition for use in automatic dishwash (ADW), for example, may include 0.0001%-50%, such as 0.001%-30%, such as 0.01%-20%, such as 0.5-15% of enzyme protein by weight of the composition.

A composition for use in laundry granulation, for example, may include 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

A composition for use in laundry liquid, for example, may include 0.0001%-10%, such as 0.001-7%, such as 0.1%-5% of enzyme protein by weight of the composition.

The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92/19709 and WO 92/19708 or the variants according to the invention may be stabilized using peptide aldehydes or ketones such as described in WO 2005/105826 and WO 2009/118375.

A variant of the present invention may also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from about 0.1% to 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized.

When included therein the detergent will usually contain from about 1% to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

When included therein the detergent will usually contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

When included therein the detergent will usually contain from about 0% to about 10% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

When included therein the detergent will usually contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2′,2″-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

The detergent composition may also contain 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include 2,2′,2″-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N,N,N′-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854, U.S. Pat. No. 5,977,053

Bleaching Systems

The detergent may contain 0-50% by weight, such as about 0.1% to about 25%, of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone®, and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator. The term bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides. Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in WO98/17767. A particular family of bleach activators of interest was disclosed in EP624154 and particularly preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like triacetin has the advantage that it is environmental friendly as it eventually degrades into citric acid and alcohol. Furthermore acetyl triethyl citrate and triacetin has a good hydrolytical stability in the product upon storage and it is an efficient bleach activator. Finally ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may also include a bleach catalyst. In some embodiments the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R¹ is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R¹ is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259 and WO2007/087242. Suitable photobleaches may for example be sulfonated zinc phthalocyanine

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % to about 0.04 wt % fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and WO2007/087243.

Additional Enzymes

The detergent additive as well as the detergent composition may comprise one or more (additional) enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Cellulases

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Example of cellulases exhibiting endo-beta-1,4-glucanase activity (EC 3.2.1.4) are those having described in WO02/099091.

Other examples of cellulases include the family 45 cellulases described in WO96/29397, and especially variants thereof having substitution, insertion and/or deletion at one or more of the positions corresponding to the following positions in SEQ ID NO: 8 of WO 02/099091: 2, 4, 7, 8, 10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113, 114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146, 147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178, 181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20, preferably selected among P19A, G20K, Q44K, N48E, Q119H or Q146 R.

Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Proteases

Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4 or other metalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140). Other useful proteases may be those described in WO92/175177, WO01/016285, WO02/026024 and WO02/016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270, WO94/25583 and WO05/040372, and the chymotrypsin proteases derived from Cellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO95/23221, and variants thereof which are described in WO92/21760, WO95/23221, EP1921147 and EP1921148.

Examples of metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int.) such as those derived from Bacillus amyloliquefaciens. Examples of useful proteases are the variants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263, WO11/036264, especially the variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred the subtilase variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipase from Thermobifida fusca (WO11/084412), Geobacillus stearothermophilus lipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (WO12/137147).

Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143), acyltransferase from Mycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Amylases

Suitable amylases which can be used together with a subtilase variant of the invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 3 in WO 95/10603 or variants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90% sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T491+G107A+H156Y+A181T+N190F+I201F+A209V+Q2645.

Further amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variants are those having a deletion in positions 181 and 182 or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90% sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181 or of T182 and/or G183. Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A/S), and Rapidase™, Purastar™/Effectenz™, Powerase and Preferenz S100 (from Genencor International Inc./DuPont).

Peroxidases/Oxidases

Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

Dispersants: The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents: The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the composition.

Fluorescent whitening agent: The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0,01% to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate; 4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate, 4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate; 4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and 2-(stilbyl-4″-naptho-1.,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino)stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins. Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.

Soil release polymers: The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference). Furthermore random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.

Anti-redeposition agents: The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.

Formulation of Detergent Products

The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. There are a number of detergent formulation forms such as layers (same or different phases), pouches, as well as forms for machine dosing unit.

Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blend compositions comprising hydrolytically degradable and water soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry detergent composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film. The compartment for liquid components can be different in composition than compartments containing solids. Ref: (US2009/0011970 A1).

Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

A liquid or gel detergent, which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.

Laundry Soap Bars

The enzymes of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.

The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺ and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.

The laundry soap bar may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g. a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. The premix of the invention may be added to the soap at different stages of the process. For example, the premix containing a soap, an enzyme, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and the mixture is then plodded. The enzyme and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO09/092699, EP1705241, EP1382668, WO07/001262, U.S. Pat. No. 6,472,364, WO04/074419 or WO09/102854. Other useful detergent formulations are described in WO09/124162, WO09/124163, WO09/117340, WO09/117341, WO09/117342, WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/112296, WO09/112298, WO09/103822, WO09/087033, WO09/050026, WO09/047125, WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375, WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545, WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/115391, WO09/115392, WO09/074398, WO09/074403, WO09/068501, WO09/065770, WO09/021813, WO09/030632, and WO09/015951.

WO2011025615, WO2011016958, WO2011005803, WO2011005623, WO2011005730, WO2011005844, WO2011005904, WO2011005630, WO2011005830, WO2011005912, WO2011005905, WO2011005910, WO2011005813, WO2010135238, WO2010120863, WO2010108002, WO2010111365, WO2010108000, WO2010107635, WO2010090915, WO2010033976, WO2010033746, WO2010033747, WO2010033897, WO2010033979, WO2010030540, WO2010030541, WO2010030539, WO2010024467, WO2010024469, WO2010024470, WO2010025161, WO2010014395, WO2010044905,

WO2010145887, WO2010142503, WO2010122051, WO2010102861, WO2010099997, WO2010084039, WO2010076292, WO2010069742, WO2010069718, WO2010069957, WO2010057784, WO2010054986, WO2010018043, WO2010003783, WO2010003792,

WO2011023716, WO2010142539, WO2010118959, WO2010115813, WO2010105942, WO2010105961, WO2010105962, WO2010094356, WO2010084203, WO2010078979, WO2010072456, WO2010069905, WO2010076165, WO2010072603, WO2010066486, WO2010066631, WO2010066632, WO2010063689, WO2010060821, WO2010049187, WO2010031607, WO2010000636.

Uses

The present invention is also directed to methods for using the variants according to the invention or compositions thereof in laundering of textile and fabrics, such as house hold laundry washing and industrial laundry washing.

The invention is also directed to methods for using the variants according to the invention or compositions thereof in cleaning hard surfaces such as floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash).

The subtilisin variants of the present invention may be added to and thus become a component of a detergent composition. Thus one aspect of the invention relates to the use of a subtilisin variant comprising the double substitution 194P+261D and optionally one or more alterations selected from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 in a cleaning process such as laundering and/or hard surface cleaning.

In a further aspect, the invention relates to the use of a subtilisin variant comprising the double substitution A194P+N261D and optionally one or more alterations selected from the group consisting of S3{I, L, P, V, Y}, S9{A, G, M, T}, N18{A, G, M, S, T}, P40{D, E}, N43{D, E}, R45{D, E}, T58{F, Y}, Q59{D, E}, G61{D, E}, 172{L, V}, N76{D, E}, S99{A, E, G, M, T}, V104{F, Y}, S105{D, E}, H120{D, E, I, L, V}, S130*, A133{D, E}, S141{F, Y}, Q182{D, E}, N183{D, E}, N184{D, E}, V205{I, L}, Q206{D, E}, S212E, N218{D, E}, T224{A, G, M, S}, P225{A, G, M, S, T}, A228{G, M, S, T}, Q236{D, E}, Q245{H, K}, 5259{D, E} and L262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 wherein the variant has a sequence identity of at least 60%, such as at least 65%, such as at least 70%, e.g., at least 75%, at least 76% at least 77% at least 78% at least 79% at least 80%, at least 81% at least 82% at least 83% at least 84% at least 85%, at least 86% at least 87% at least 88% at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, or at least 98%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 4 in a cleaning process such as laundering and/or hard surface cleaning.

Thus one aspect of the invention relates to the use of a subtilisin variant comprising the double substitution 194P+261D and optionally one or more alterations selected from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 in a cleaning process such as laundering and/or hard surface cleaning and wherein the subtilase variant has improved stability, in particular improved in wash stability, relative to the parent or relative to a protease parent having the identical amino acid sequence of said variant but not having the alterations at one or more of said positions when measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

In a further aspect, the invention relates to the use of a subtilisin variant comprising the double substitution A194P+N261D and optionally one or more alterations selected from the group consisting of S3{I, L, P, V, Y}, S9{A, G, M, T}, N18{A, G, M, S, T}, P40{D, E}, N43{D, E}, R45{D, E}, T58{F, Y}, Q59{D, E}, G61{D, E}, 172{L, V}, N76{D, E}, S99{A, E, G, M, T}, V104{F, Y}, S105{D, E}, H120{D, E, I, L, V}, S130*, A133{D, E}, S141{F, Y}, Q182{D, E}, N183{D, E}, N184{D, E}, V205{I, L}, Q206{D, E}, S212E, N218{D, E}, T224{A, G, M, S}, P225{A, G, M, S, T}, A228{G, M, S, T}, Q236{D, E}, Q245{H, K}, S259{D, E} and L262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 wherein the variant has a sequence identity of at least 60%, such as at least 65%, such as at least 70%, e.g., at least 75%, at least 76% at least 77% at least 78% at least 79% at least 80%, at least 81% at least 82% at least 83% at least 84% at least 85%, at least 86% at least 87% at least 88% at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, or at least 98%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 4 in a cleaning process such as laundering and/or hard surface cleaning and wherein the subtilase variant has improved stability, in particular improved in wash stability, relative to the mature polypeptide of SEQ ID NO: 4 when measured using the ‘in wash stability assay’ as described in the Materials and Methods section herein.

The subtilisin variants of the present invention may be added to and thus become a component of a detergent composition. Thus one aspect of the invention relates to the use of a subtilisin variant comprising the double substitution 194P+261D and optionally one or more alterations selected from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 in a cleaning process such as laundering and/or hard surface cleaning and wherein the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance relative to the parent or relative to a protease parent having the identical amino acid sequence of said variant but not having the alterations at one or more of said positions when measured using the ‘in wash stability assay’ and the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash respectively as described in the Materials and Methods section herein.

In a further aspect, the invention relates to the use of a subtilisin variant comprising the double substitution A194P+N261D and optionally one or more alterations selected from the group consisting of S3{I, L, P, V, Y}, S9{A, G, M, T}, N18{A, G, M, S, T}, P40{D, E}, N43{D, E}, R45{D, E}, T58{F, Y}, Q59{D, E}, G61{D, E}, 172{L, V}, N76{D, E}, S99{A, E, G, M, T}, V104{F, Y}, S105{D, E}, H120{D, E, 1, L, V}, S130*, A133{D, E}, S141{F, Y}, Q182{D, E}, N183{D, E}, N184{D, E}, V205{I, L}, Q206{D, E}, S212E, N218{D, E}, T224{A, G, M, S}, P225{A, G, M, S, T}, A228{G, M, S, T}, Q236{D, E}, Q245{H, K}, S259{D, E} and L262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 wherein the variant has a sequence identity of at least 60%, such as at least 65%, such as at least 70%, e.g., at least 75%, at least 76% at least 77% at least 78% at least 79% at least 80%, at least 81% at least 82% at least 83% at least 84% at least 85%, at least 86% at least 87% at least 88% at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, or at least 98%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 4 in a cleaning process such as laundering and/or hard surface cleaning and wherein the subtilase variant has improved stability, in particular improved in wash stability, and on par or improved wash performance relative to the mature polypeptide of SEQ ID NO: 4 when measured using the ‘In Wash Stability Assay’ and the Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash respectively as described in the Materials and Methods section herein.

A preferred aspect of the invention relates to the use of a subtilisin variant comprising the double substitution A194P+N261D and optionally one or more alterations selected from the group consisting of 3V, 3Y, 9M, 18S, 18T, 18D, 40E, 43D, 43E, 45E, 58Y, 59E, 61D, 72V, 76D, 99D, 104Y, 1054D, 120I, 120D, 130*, 133E, 141F, 182E, 183D, 184D, 205I, 212D, 218D, 228S, 236E and 262Y, wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 wherein the variant has a sequence identity of at least 60%, such as at least 65%, such as at least 70%, e.g., at least 75%, at least 76% at least 77% at least 78% at least 79% at least 80%, at least 81% at least 82% at least 83% at least 84% at least 85%, at least 86% at least 87% at least 88% at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, or at least 98%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 4 in a cleaning process such as laundering and/or hard surface cleaning.

A detergent composition of the present invention may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the present invention provides a detergent additive comprising a polypeptide of the present invention as described herein.

The cleaning process or the textile care process may for example be a laundry process, a dishwashing process or cleaning of hard surfaces such as bathroom tiles, floors, table tops, drains, sinks and washbasins. Laundry processes can for example be household laundering, but it may also be industrial laundering. Furthermore, the invention relates to a process for laundering of fabrics and/or garments where the process comprises treating fabrics with a washing solution containing a detergent composition, and at least one protease variant of the invention. The cleaning process or a textile care process can for example be carried out in a machine washing process or in a manual washing process. The washing solution can for example be an aqueous washing solution containing a detergent composition.

The last few years there has been an increasing interest in replacing components in detergents, which is derived from petrochemicals with renewable biological components such as enzymes and polypeptides without compromising the wash performance. When the components of detergent compositions change new enzyme activities or new enzymes having alternative and/or improved properties compared to the common used detergent enzymes such as proteases, lipases and amylases are needed to achieve a similar or improved wash performance when compared to the traditional detergent compositions.

The invention further concerns the use of subtilase variants of the invention in a proteinaceous stain removing processes. The proteinaceous stains may be stains such as food stains, e.g., baby food, sebum, cocoa, egg, blood, milk, ink, grass, or a combination hereof.

Typical detergent compositions include various components in addition to the enzymes, these components have different effects, some components like the surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away, other components like bleach systems remove discolor often by oxidation and many bleaches also have strong bactericidal properties, and are used for disinfecting and sterilizing. Yet other components like builder and chelator softens, e.g., the wash water by removing the metal ions form the liquid.

In a particular embodiment, the invention concerns the use of a composition comprising a subtilase variant of the invention and one or more detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

In a particular embodiment, the invention concerns the use of a composition comprising a subtilase variant of the invention and one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof.

In a particular embodiment, the invention concerns the use of a composition comprising a subtilase variant of the invention, one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof and one or more detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

Washing Method

The present invention relates to a method of cleaning a fabric, a dishware or hard surface with a detergent composition comprising a protease variant of the invention.

A preferred embodiment concerns a method of cleaning, said method comprising the steps of: contacting an object with a detergent composition comprising a protease variant of the invention under conditions suitable for cleaning said object. In a preferred embodiment the detergent composition is used in a laundry or a dish wash process.

Still another embodiment relates to a method for removing stains from fabric or dishware which comprises contacting said fabric or dishware with a composition comprising a protease of the invention under conditions suitable for cleaning said object.

Also contemplated are compositions and methods of treating fabrics (e.g., to desize a textile) using one or more of the protease of the invention. The protease can be used in any fabric-treating method which is well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, in one aspect, the feel and appearance of a fabric is improved by a method comprising contacting the fabric with a protease in a solution. In one aspect, the fabric is treated with the solution under pressure.

The detergent compositions of the present invention are suited for use in laundry and hard surface applications, including dish wash. Accordingly, the present invention includes a method for laundering a fabric or washing dishware. The method comprises the steps of contacting the fabric/dishware to be cleaned with a solution comprising the detergent composition according to the invention. The fabric may comprise any fabric capable of being laundered in normal consumer use conditions. The dishware may comprise any dishware such as crockery, cutlery, ceramics, plastics such as melamine, metals, china, glass and acrylics. The solution preferably has a pH from about 5.5 to about 11.5. The compositions may be employed at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. The water temperatures typically range from about 5° C. to about 95° C., including about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 40° C., about 45° C., about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., about 75° C., about 80° C., about 85° C. and about 90° C. The water to fabric ratio is typically from about 1:1 to about 30:1.

Variations in local and regional conditions, such as water hardness and wash temperature call for regional detergent compositions. Table 4 provide ranges for the composition of a typical European automatic dish wash (ADW) detergent.

TABLE 4 Typical European ADW detergent composition P-Containing formulation P-Free formulations 20-50% STPP 10-20% Na Citrate (or Chelating agent) 15-45% Soda (sodium carbonate) 25% Soda (sodium carbonate) 5-15% Sodium percabonate 5-10% Sodium percabonate 0-20% Sodium disilicate 5-25% Sodium disilicate 2-3% TAED 0-3% TAED 2-6% Polymers 2-6% Polymers 1-2% Phosphonate 1-20% Sodium sulfate 3-5% Surfactants <5% Surfactants <5% Enzymes <5% Enzymes To 100% Rest (perfume, dye, To 100% Rest (perfume, dye, corrosion inhibitor. etc.) pH 9-11 corrosion inhibitor. etc.) pH 9-11

The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents and protease inhibitors, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, different salts such as NaCl; KCl; lactic acid, formic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, or a peptide aldehyde such as di-, tri- or tetrapeptide aldehydes or aldehyde analogues (either of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2, or CH2X (X=halogen), B0 is a single amino acid residue (preferably with an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably one, two or three), optionally comprising an N-terminal protection group, or as described in WO09118375, WO98/13459) or a protease inhibitor of the protein type such as RASI, BASI, WASI (bifunctional alpha-amylase/subtilisin inhibitors of rice, barley and wheat) or 012 or SSI. The composition may be formulated as described in e.g. WO 92/19709, WO 92/19708 and U.S. Pat. No. 6,472,364. In some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)).

In some preferred embodiments, the detergent compositions provided herein are typically formulated such that, during use in aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 11.5, or in alternative embodiments, even from about 6.0 to about 10.5. In some preferred embodiments, granular or liquid laundry products are formulated to have a pH from about 6 to about 8. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art. The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Protease Assay (Suc-AAPF-pNA Assay)

-   pNA substrate: Suc-AAPF-pNA (Bachem L-1400). -   Temperature: Room temperature (25° C.) -   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100     mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 9.0.

20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% Triton X-100). The increase in OD₄₀₅ was monitored as a measure of the protease activity.

In Wash Stability Assay

In wash stability is measured using the two model detergents as defined in table 5 below.

TABLE 5 Composition of the MGDA and STPP model detergents MGDA model STPP model Component detergent detergent MGDA (40% solution) 1.67 g/l STPP 1.65 g/l Sodium carbonate 0.66 g/l 0.66 g/l Sodium percarbonate (Dream) 0.33 g/l 0.33 g/l Sodium disilicate 0.17 g/l 0.17 g/l TAED (Dream) 0.10 g/l 0.10 g/l Sokalan CP5 (39.5%) 0.42 g/l 0.42 g/l Surfac 23-6.5 (100%) 0.17 g/l 0.17 g/l Sodium sulphate 1.06 g/l Phosphonate (tetrasodium HEDP) 0.07 g/l CaCl₂ 3 mM 3 mM MgCl₂ 0.75 mM 0.75 mM NaHCO₃ 7.5 mM 7.5 mM pH 10.0 10.0

Both detergents are dissolved in 50 mM CHES buffer to ensure that pH is maintained during the experiment at 10.0 also after addition of protease sample.

Protease culture supernatants are pre-diluted 2-4 times and purified protease samples are diluted to approximately 0.1 and 0.05 mg/ml using deionized water. 10 μl diluted protease sample is then mixed with 190 μl model detergent solution in a well of a 0.2 ml 96-well PCR plate. After mixing, 20 μl is transferred to a 96-well microtiter plate (Nunc F) and initial protease activity is measured by adding 100 μl Suc-AAPF-pNA substrate solution (0.72 mg/ml Suc-Ala-Ala-Pro-Phe-pNA (Bachem L-1400) in 0.1 M Tris, pH 8.6) to each well, mixing and measuring absorbance at 405 nm every 20 s for 5 min on a SpectraMax Plus (Molecular Devices). Slope from linear regression on initial absorbance measurements is used for activity calculations.

The proteases in the PCR plate are then stressed by 30 min incubation at 58° C. for STPP model detergent and 60 or 62° C. for MGDA model detergent in a BioRad T100 Thermal Cycler. After rapid cooling to room temperature, 20 μl is transferred to a 96 well microtiter plate and residual activity is measured as described for the initial protease activity. The temperatures in the stress step are chosen to give suitable residual activities of the Savinase reference (mature polypeptide of SEQ ID NO: 4) and the variants (preferably in the interval 10 to 80% of the initial activity).

The decrease in activity during the stress step is assumed to be exponential. Thus, the half-life during the stress step is calculated using the formula:

T½=T*ln(2)/ln(A(Initial)/A(Residual))

where T½ is the half-life, T is the incubation time (30 min), A(Initial) is the initial protease activity, and A(Residual) is the protease activity after the stress step. All protease samples are tested twice (using 2 times the same sample dilution for culture supernatants and 0.1 and 0.05 mg/ml for purified protease samples). Relative in wash stability improvement factor is then calculated by:

Relative In Wash Stability Improvement Factor=Avg(T½(Sample))/Avg(T½(Reference)

where Avg(T½(Samples)) is the average of the half-lifes for the given protease sample and Avg(T½(Reference)) is the average of the half-lifes for the Savinase reference sample (mature polypeptide of SEQ ID NO: 4).

Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash

Washing experiments are performed in order to assess the wash performance of selected protease variants in dish wash detergent compositions. The proteases of the present application are tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA, the wash performance of many small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid that firmly squeezes the melamine tile to be washed against the slot openings. During the wash, the plate, test solutions, melamine tile and lid are vigorously shaken to bring the test solution in contact with the soiled melamine tile and apply mechanical stress in a regular, periodic oscillating manner. For further description see WO 02/42740 especially the paragraph “Special method embodiments” at page 23-24.

The experiment is conducted under the experimental conditions as specified in tables 6 and 7 below.

TABLE 6 AMSA Experimental Conditions using ADW model detergent with MGDA ADW model detergent with MGDA As defined in table 5 Detergent dosage 3.33 g/L Test solution volume 160 micro L pH 10.0 Wash time 20 minutes Temperature 45° C. Water hardness 21 °dH Enzyme concentration in test solution 5.3, 10.7 mg enzyme protein/liter Test material Egg yolk melamine tile (DM-21)

TABLE 7 AMSA Experimental Conditions using ADW model detergent with STPP ADW model detergent with STPP As defined in table 5 Detergent dosage 3.33 g/L Test solution volume 160 micro L pH 10.0 Wash time 20 minutes Temperature 45° C. Water hardness 21°dH Enzyme concentration in test solution 5.3, 10.7 mg enzyme protein/liter Test material Egg yolk melamine tile (DM-21)

Water hardness is adjusted to 21° dH by addition of CaCl₂, MgCl₂, and NaHCO₃ (Ca²⁺:Mg²⁺:CO₃ ²⁻=4:1:10) to the test system. After washing the egg yolk melamine tiles are flushed in tap water and dried.

The performance of the enzyme variant is measured as the brightness of the colour of the melamine tile washed with that specific protease. Brightness can also be expressed as the intensity of the light reflected from the sample when illuminated with white light. When the sample is stained the intensity of the reflected light is lower, than that of a clean sample. Therefore the intensity of the reflected light can be used to measure wash performance of a protease. Color measurements were made with a professional flatbed scanner (EPSON EXPRESSION 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark), which is used to capture an image of the washed melamine tiles.

To extract a value for the light intensity from the scanned images, a special designed software application is used (Novozymes Colour Vector Analyzer). The program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:

Int=√{square root over (r ² +g ² +b ²)}.

Textiles

Standard egg yolk melamine tiles (DM-21) were obtained from Center For Test materials BV, P.O. Box 120, 3133 KT Vlaardingen, the Netherlands.

Example 1 Preparation and Expression of Variants

The following summarizes the mutation and introduction of an expression cassette into Bacillus subtilis. All DNA manipulations were done by PCR (e.g. Sambrook et al.; Molecular Cloning; Cold Spring Harbor Laboratory Press) and can be repeated by everybody skilled in the art.

Recombinant B. subtilis constructs encoding subtilase variants were used to inoculate shakeflasks containing a rich media (e.g. PS-1: 100 g/L Sucrose (Danisco cat. no. 109-0429), 40 g/L crust soy (soy bean flour), 10 g/L Na2HPO4.12H20 (Merck cat. no. 6579), 0.1 ml/L replace-Dowfax63N10 (Dow). Cultivation typically takes 4 days at 30° C. shaking with 220 rpm.

Example 2 Fermentation of Variants

Fermentation may be performed by methods well known in the art or as follows. A B. subtilis strain harboring the relevant expression plasmid was streaked on a LB agar plate, and grown overnight at 37° C. The colonies were transferred to 100 ml PS-1 media in a 500 ml shaking flask. Cells and other undissolved material were removed from the fermentation broth by centrifugation at 4500 rpm for 20-25 minutes. Afterwards the supernatant was filtered to obtain a clear solution.

Example 3 Purification of Variants

The culture broth was centrifuged (26000×g, 20 min) and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filtration unit in order to remove the rest of the Bacillus host cells. pH in the 0.2 μm filtrate was adjusted to pH 8 with 3M Tris base and the pH adjusted filtrate was applied to a MEP Hypercel column (from Pall corporation) equilibrated in 20 mM Tris/HCl, 1 mM CaCl₂, pH 8.0. After washing the column with the equilibration buffer, the column was step-eluted with 20 mM CH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5. Fractions from the column were analysed for protease activity (using the Suc-AAPF-pNA assay at pH 9) and peak-fractions were pooled. The pH of the pool from the MEP Hypercel column was adjusted to pH 6 with 20% (v/v) CH₃COOH or 3M Tris base and the pH adjusted pool was diluted with deionized water to the same conductivity as 20 mM MES/NaOH, 2 mM CaCl₂, pH 6.0. The diluted pool was applied to a SP-sepharose FF column (from GE Healthcare) equilibrated in 20 mM MES/NaOH, 2 mM CaCl₂, pH 6.0. After washing the column with the equilibration buffer, the protease was eluted with a linear NaCl gradient (0-->0.5M) in the same buffer over five column volumes. Fractions from the column were analysed for protease activity (using the Suc-AAPF-pNA assay at pH 9) and active fractions were analysed by SDS-PAGE. The fractions, where only one band was seen on the coomassie stained SDS-PAGE gel, were pooled as the purified preparation and was used for further experiments.

Example 4 In Wash Stability

In wash stability was measured according to the ‘In wash stability assay’ as described herein using the MGDA and STPP model detergents as defined in table 4, wherein the mutations were carried out on the Savinase (mature polypeptide of SEQ ID NO: 4) backbone. The results are presented in table 8.

TABLE 8 In Wash Stability Data using MGDA and STPP model detergents Relative Half Life (T½) Mutations STPP MGDA A194P + N261D 1.86 1.71 S3V + A194P + N261D 4.03 4.15 N43E + A194P + N261D 3.89 2.56 Q182E + A194P + N261D 3.69 4.54 N76D + A194P + N261D 3.56 5.14 A194P + V205I + N261D 3.53 3.65 S3Y + A194P + N261D 3.51 3.80 N43D + A194P + N261D 2.95 2.58 A194P + S212D + N261D 2.90 3.14 A194P + N261D + L262Y 2.76 2.40 N18S + A194P + N261D 2.59 2.76 H120I + A194P + N261D 2.53 2.10 S130* + A194P + N261D 2.41 2.07 A194P + N218D + N261D 2.41 2.22 P40E + A194P + N261D 2.36 2.26 S9M + A194P + N261D 2.33 2.41 A194P + Q236E + N261D 2.20 2.13 A194P + A228S + N261D 2.15 2.05 N18T + A194P + N261D 2.08 1.74 N18D + A194P + N261D 2.07 1.84 Q59E + A194P + N261D 2.00 2.13 G61D + A194P + L250Q + N261D 1.99 1.77 I72V + A194P + N261D 1.88 2.18 S105D + A194P + N261D 1.83 1.75 V104Y + A194P + N261D 1.83 1.60 S99D + A194P + N261D 1.79 2.44 R45E + A194P + N261D 1.78 1.92 S141F + A194P + N261D 1.71 1.98 T58Y + A194P + N261D 1.70 1.54 H120D + A194P + N261D 1.68 1.87 A133E + A194P + N261D 1.68 1.63 N183D + A194P + N261D 1.67 1.65 N184D + A194P + N261D 1.62 1.59

PREFERRED EMBODIMENTS

The following preferred embodiments further describe the invention.

Embodiment 1

A subtilase variant having protease activity, comprising the double substitution 194P+261D and one or more alterations from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2.

Embodiment 2

The variant of embodiment 1, wherein the double substitution is A194P+N261D.

Embodiment 3

The variant of any of embodiments 1 or 2 wherein the variant subtilase is:

-   -   a) a polypeptide that has at least 60%, e.g., at least 65%, at         least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 95%, at least 96%, at least 97%, at least 98% but         less than 100% sequence identity to the mature polypeptide of         the parent subtilase;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with         -   (i) the mature polypeptide coding sequence of parent             subtilase or         -   (ii) the full-length complement of (i); or     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60%, e.g., at least 65%, at least 70%, at least 75%, at         least 80%, at least 85%, at least 90%, at least 95%, at least         96%, at least 97%, at least 98% but less than 100% sequence         identity to the mature polypeptide coding sequence of parent         subtilase.

Embodiment 4

The variant of any of embodiments 1 or 2 wherein the variant subtilase is:

-   -   a) a polypeptide that has at least 60%, e.g., at least 65%, at         least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 95%, at least 96%, at least 97%, at least 98% but         less than 100% sequence identity to the mature polypeptide of         SEQ ID NO: 4;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with         -   (i) the mature polypeptide coding sequence of SEQ ID NO: 3             or         -   (ii) the full-length complement of (i); or     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60%, e.g., at least 65%, at least 70%, at least 75%, at         least 80%, at least 85%, at least 90%, at least 95%, at least         96%, at least 97%, at least 98% but less than 100% sequence         identity to the mature polypeptide coding sequence of SEQ ID NO:         3.

Embodiment 5

The variant of any of embodiments 1-4, wherein the total number of alterations is between 3 and 30, preferably between 3 and 20, more preferably between 3 and 15, even more preferably between 3 and 10, most preferably between 3 and 8 alterations.

Embodiment 6

The variant of any of embodiments 1-5, wherein the total number of alterations is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 alterations.

Embodiment 7

The variant of any of embodiments 1-6, wherein the variant consists of 150 to 350, e.g., 175 to 330, 200 to 310, 220 to 300, 240 to 290, 260 to 280 or 269 to 275 amino acids.

Embodiment 8

The variant of any of embodiments 1-7, wherein the variant comprises

one or more of the alterations selected from the group consisting of:

3F+194P+261D;

3I+194P+261D;

3L+194P+261D;

3V+194P+261D;

3Y+194P+261D;

9A+194P+261D;

9G+194P+261D;

9M+194P+261D;

9T+194P+261D;

9+194P+261D;

18A+194P+261D;

18D+194P+261D;

18E+194P+261D;

18G+194P+261D;

18M+194P+261D;

18S+194P+261D;

18T+194P+261D;

40D+194P+261D;

40E+194P+261D;

43D+194P+261D;

43E+194P+261D;

45D+194P+261D;

45E+194P+261D;

58F+194P+261D;

58Y+194P+261D;

59D+194P+261D;

59E+194P+261D;

61D+194P+261D;

61E+194P+261D;

72L+194P+261D;

72V+194P+261D;

76D+194P+261D;

76E+194P+261D;

99A+194P+261D;

99D+194P+261D;

99E+194P+261D;

99M+194P+261D;

99T+194P+261D;

104F+194P+261D;

104Y+194P+261D;

105D+194P+261D;

105E+194P+261D;

120D+194P+261D;

120E+194P+261D;

120I+194P+261D;

120L+194P+261D;

120V+194P+261D;

130*194P+261D;

133D+194P+261D;

133E+194P+261D;

141F+194P+261D;

141Y+194P+261D;

182D+194P+261D;

182E+194P+261D;

183D+194P+261D;

183E+194P+261D;

184D+194P+261D;

184E+194P+261D;

194P+205I+261D;

194P+205L+261D;

194P+206D+261D;

194P+206E+261D;

194P+212D+261D;

194P+212E+261D;

194P+218D+261D;

194P+218E+261D;

194P+224A+261D;

194P+224G+261D;

194P+224M+261D;

194P+224S+261D;

194P+225A+261D;

194P+225G+261D;

194P+225M+261D;

194P+225S+261D;

194P+225T+261D;

194P+228G+261D;

194P+228M+261D;

194P+228S+261D;

194P+228T+261D;

194P+236D+261D;

194P+236E+261D;

194P+245H+261D;

194P+245K+261D;

194P+259D+261D;

194P+259E+261D;

194P+261D+262F;

194P+261D+262W; and

194P+261D+262Y.

Embodiment 9

The variant of any of embodiments 1-8, comprising a substitution, deletion and/or insertion at one or more positions (e.g. several) selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270 wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2.

Embodiment 10

The variant of any of embodiments 1-9, which has an improved stability, preferably improved in wash stability, compared to the parent or compared to a reference protease.

Embodiment 11

The variant of embodiment 10, which has an improved wash performance compared to the parent or compared to a reference protease.

Embodiment 12

A composition comprising a variant according to any of embodiments 1 to 11.

Embodiment 13

The detergent composition of embodiment 12 further comprising one or more detergent components.

Embodiment 14

The composition according to any of embodiment 12-13 further comprising one or more additional enzymes selected from the group comprising of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof.

Embodiment 15

The composition according to any of embodiments 12-14 in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

Embodiment 16

Use of the composition according to any of embodiments 12-15 in a cleaning process, such as laundry or hard surface cleaning such as dish wash.

Embodiment 17

A method for obtaining a subtilase variant, comprising (a) introducing into a parent subtilase the double substitution 194P+261D and one or more alterations from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y} wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2 and (b) recovering the variant.

Embodiment 18

The method of embodiment 17, wherein the subtilase variant is selected from the list consisting of:

-   -   a) a polypeptide that has at least 60%, e.g., at least 65%, at         least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 95%, at least 96%, at least 97%, at least 98% but         less than 100% sequence identity to the mature polypeptide of         the parent subtilase;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with         -   (i) the mature polypeptide coding sequence of the parent             subtilase or         -   (ii) the full-length complement of (i); and     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60%, e.g., at least 65%, at least 70%, at least 75%, at         least 80%, at least 85%, at least 90%, at least 95%, at least         96%, at least 97%, at least 98% but less than 100% sequence         identity to the mature polypeptide coding sequence of the parent         subtilase.

Embodiment 19

The method of embodiment 17, wherein the double substitution is A194P+N261D and the subtilase variant is selected from the list consisting of:

-   -   a) a polypeptide that has at least 60%, e.g., at least 65%, at         least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 95%, at least 96%, at least 97%, at least 98% but         less than 100% sequence identity to the mature polypeptide of         SEQ ID NO: 4;     -   b) a polypeptide that is encoded by a polynucleotide that         hybridizes under low stringency conditions, medium stringency         conditions, medium-high stringency conditions, high stringency         conditions, or very high stringency conditions with         -   (i) the mature polypeptide coding sequence of SEQ ID NO: 3             or         -   (ii) the full-length complement of (i); and     -   c) a polypeptide that is encoded by a polynucleotide having at         least 60%, e.g., at least 65%, at least 70%, at least 75%, at         least 80%, at least 85%, at least 90%, at least 95%, at least         96%, at least 97%, at least 98% but less than 100% sequence         identity to the mature polypeptide coding sequence of SEQ ID NO:         3.

Embodiment 20

The method of any of embodiments 17-19, wherein one or more of the alterations introduced into a parent subtilase is selected from the group consisting of:

3F+194P+261D;

3I+194P+261D;

3L+194P+261D;

3V+194P+261D;

3Y+194P+261D;

9A+194P+261D;

9G+194P+261D;

9M+194P+261D;

9T+194P+261D;

9+194P+261D;

18A+194P+261D;

18D+194P+261D;

18E+194P+261D;

18G+194P+261D;

18M+194P+261D;

18S+194P+261D;

18T+194P+261D;

40D+194P+261D;

40E+194P+261D;

43D+194P+261D;

43E+194P+261D;

45D+194P+261D;

45E+194P+261D;

58F+194P+261D;

58Y+194P+261D;

59D+194P+261D;

59E+194P+261D;

61D+194P+261D;

61E+194P+261D;

72L+194P+261D;

72V+194P+261D;

76D+194P+261D;

76E+194P+261D;

99A+194P+261D;

99D+194P+261D;

99E+194P+261D;

99M+194P+261D;

99T+194P+261D;

104F+194P+261D;

104Y+194P+261D;

105D+194P+261D;

105E+194P+261D;

120D+194P+261D;

120E+194P+261D;

120I+194P+261D;

120L+194P+261D;

120V+194P+261D;

130*+194P+261D;

133D+194P+261D;

133E+194P+261D;

141F+194P+261D;

141Y+194P+261D;

182D+194P+261D;

182E+194P+261D;

183D+194P+261D;

183E+194P+261D;

184D+194P+261D;

184E+194P+261D;

194P+205I+261D;

194P+205L+261D;

194P+206D+261D;

194P+206E+261D;

194P+212D+261D;

194P+212E+261D;

194P+218D+261D;

194P+218E+261D;

194P+224A+261D;

194P+224G+261D;

194P+224M+261D;

194P+224S+261D;

194P+225A+261D;

194P+225G+261D;

194P+225M+261D;

194P+225S+261D;

194P+225T+261D;

194P+228G+261D;

194P+228M+261D;

194P+228S+261D;

194P+228T+261D;

194P+236D+261D;

194P+236E+261D;

194P+245H+261D;

194P+245K+261D;

194P+259D+261D;

194P+259E+261D;

194P+261D+262F;

194P+261D+262W; and

194P+261D+262Y.

Embodiment 21

The method of any of embodiments 18-22, wherein further alterations introduced into a parent subtilase comprises a substitution, deletion and/or insertion at one or more positions (e.g. several) selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270 wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2.

Embodiment 22

A method for removing a stain from a surface which comprises contacting the surface with a composition according to any of embodiments 12 to 15. 

1. A subtilase variant having protease activity and comprises the double substitution 194P+261D and wherein the positions correspond to the positions of the mature polypeptide of SEQ ID NO:
 2. 2. The variant according to claim 1 which further comprises one or more alterations from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y}.
 3. The variant of claim 1, wherein the double substitution is A194P+N261D.
 4. The variant of claim 1 wherein the variant subtilase is: a) a polypeptide that has at least 60% but less than 100% sequence identity to the mature polypeptide of the parent subtilase; b) a polypeptide that is encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of parent subtilase or (ii) the full-length complement of (i); or c) a polypeptide that is encoded by a polynucleotide having at least 60% but less than 100% sequence identity to the mature polypeptide coding sequence of parent subtilase.
 5. The variant of claim 1 wherein the variant subtilase is: a) a polypeptide that has at least 60% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4; b) a polypeptide that is encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 3 or (ii) the full-length complement of (i); or c) a polypeptide that is encoded by a polynucleotide having at least 60% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:
 3. 6. The variant of claim 1, wherein the total number of alterations is between 3 and 30 alterations.
 7. (canceled)
 8. The variant of claim 1, wherein the variant consists of 150 to 350 amino acids.
 9. The variant of claim 1, wherein the variant comprises one or more of the alterations selected from the group consisting of: 3F+194P+261D; 3I+194P+261D; 3L+194P+261D; 3V+194P+261D; 3Y+194P+261D; 9A+194P+261D; 9G+194P+261D; 9M+194P+261D; 9T+194P+261D; 9+194P+261D; 18A+194P+261D; 18D+194P+261D; 18E+194P+261D; 18G+194P+261D; 18M+194P+261D; 18S+194P+261D; 18T+194P+261D; 40D+194P+261D; 40E+194P+261D; 43D+194P+261D; 43E+194P+261D; 45D+194P+261D; 45E+194P+261D; 58F+194P+261D; 58Y+194P+261D; 59D+194P+261D; 59E+194P+261D; 61D+194P+261D; 61E+194P+261D; 72L+194P+261D; 72V+194P+261D; 76D+194P+261D; 76E+194P+261D; 99A+194P+261D; 99D+194P+261D; 99E+194P+261D; 99M+194P+261D; 99T+194P+261D; 104F+194P+261D; 104Y+194P+261D; 105D+194P+261D; 105E+194P+261D; 120D+194P+261D; 120E+194P+261D; 120I+194P+261D; 120L+194P+261D; 120V+194P+261D; 130*+194P+261D; 133D+194P+261D; 133E+194P+261D; 141F+194P+261D; 141Y+194P+261D; 182D+194P+261D; 182E+194P+261D; 183D+194P+261D; 183E+194P+261D; 184D+194P+261D; 184E+194P+261D; 194P+205I+261D; 194P+205L+261D; 194P+206D+261D; 194P+206E+261D; 194P+212D+261D; 194P+212E+261D; 194P+218D+261D; 194P+218E+261D; 194P+224A+261D; 194P+224G+261D; 194P+224M+261D; 194P+224S+261D; 194P+225A+261D; 194P+225G+261D; 194P+225M+261D; 194P+225S+261D; 194P+225T+261D; 194P+228G+261D; 194P+228M+261D; 194P+228S+261D; 194P+228T+261D; 194P+236D+261D; 194P+236E+261D; 194P+245H+261D; 194P+245K+261D; 194P+259D+261D; 194P+259E+261D; 194P+261 D+262F; 194P+261 D+262W; and 194P+261 D+262Y.
 10. The variant of claim 1, comprising a substitution, deletion and/or insertion at one or more positions selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270 wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO:
 2. 11. The variant of claim 1, which has an improved stability, preferably improved in wash stability, compared to the parent or compared to a reference protease.
 12. The variant of claim 11, which has an improved wash performance compared to the parent or compared to a reference protease.
 13. A detergent composition comprising a variant according to claim
 1. 14. The detergent composition of claim 13 further comprising one or more detergent components.
 15. The detergent composition according to claim 13 further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof.
 16. The detergent composition according to claim 13 in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. 17-18. (canceled) 